Novel compound useful for the treatement of degenerative and inflammatory diseases

ABSTRACT

A pyrazolopyridine compound according to Formula I, able to inhibit JAK is disclosed, as well as pharmaceutically acceptable salts, a solvate thereof, solvates of the pharmaceutically acceptable salts and biologically active metabolites thereof. The compound may be prepared as a pharmaceutical composition, and may be used for the treatment or prophylaxis of a variety of conditions in mammals including humans, and particularly, such conditions as may be associated with aberrant JAK activity, including by way of non-limiting example, allergy, inflammatory conditions, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6.

RELATED APPLICATION

The present application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Application No. 61/479,956, filed Apr. 28, 2011, thecontents of which is hereby incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a compound that is able to act as aninhibitor of JAK, a family of tyrosine kinases that are involved ininflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons. In particular, thecompound of the invention inhibits JAK1 and/or JAK2. The presentinvention also provides methods for the production of the compound ofthe invention, pharmaceutical compositions comprising the compound ofthe invention, methods for the prophylaxis and/or treatment of diseasesinvolving inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons byadministering the compound of the invention.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transducecytokine signaling from membrane receptors to STAT transcriptionfactors. Four JAK family members are described, JAK1, JAK2, JAK3 andTYK2. Upon binding of the cytokine to its receptor, JAK family membersauto- and/or transphosphorylate each other, followed by phosphorylationof STATs that then migrate to the nucleus to modulate transcription.JAK-STAT intracellular signal transduction serves the interferons, mostinterleukins, as well as a variety of cytokines and endocrine factorssuch as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. etal. (2008)).

The combination of genetic models and small molecule JAK inhibitorresearch revealed the therapeutic potential of several JAKs. JAK3 isvalidated by mouse and human genetics as an immune-suppression target(O'Shea J. et al. (2004)). JAK3 inhibitors were successfully taken intoclinical development, initially for organ transplant rejection but lateralso in other immuno-inflammatory indications such as rheumathoidarthritis (RA), psoriasis and Crohn's disease(http://clinicaltrials.gov/).

TYK2 is a potential target for immuno-inflammatory diseases, beingvalidated by human genetics and mouse knock-out studies (Levy D. andLoomis C. (2007)).

JAK1 and/or JAK2 is a target in the immuno-inflammatory disease area.JAK1 and/or JAK2 heterodimerizes with the other JAKs to transducecytokine-driven pro-inflammatory signaling. Therefore, inhibition ofJAK1 and/or JAK2 is of interest for immuno-inflammatory diseases withpathology-associated cytokines that use JAK1 and/or JAK2 signaling, suchas IL-6, IL-4, IL-5, IL-12, IL-13, IL-23, or IFNgamma, as well as forother diseases driven by JAK-mediated signal transduction.

BACKGROUND OF THE INVENTION

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of an RA therapy, therefore, is not only to slow down the diseasebut to attain remission in order to stop the joint destruction. Besidesthe severity of the disease outcome, the high prevalence of RA (˜0.8% ofadults are affected worldwide) means a high socio-economic impact. (Forreviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt(2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).

JAK1 and JAK2 are implicated in intracellular signal transduction formany cytokines and hormones. Pathologies associated with any of thesecytokines and hormones can be ameliorated by JAK1 and/or JAK2inhibitors. Hence, several allergy, inflammation and autoimmunedisorders might benefit from treatment with compounds described in thisinvention including rheumatoid arthritis, systemic lupus erythematosis,juvenile idiopathic arthritis, osteoarthritis, asthma, chronicobstructive pulmonary disease (COPD), tissue fibrosis, eosinophilicinflammation, eosophagitis, inflammatory bowel diseases (e.g. Crohn's,ulcerative colitis), transplant, graft-versus-host disease, psoriasis,myositis, psoriatic arthritis, ankylosing spondylitis, juvenileideopathic arthritis, multiple sclerosis (Kopf et al., 2010).

Osteoarthritis (also referred to as OA, or wear-and-tear arthritis) isthe most common form of arthritis and is characterized by loss ofarticular cartilage, often associated with hypertrophy of the bone andpain. For an extensive review on osteoarthritis, we refer to Wieland etal. (2005).

Osteoarthritis is difficult to treat. At present, no cure is availableand treatment focuses on relieving pain and preventing the affectedjoint from becoming deformed. Common treatments include the use ofnon-steroidal anti-inflammatory drugs (NSAIDs). Although dietarysupplements such as chondroitin and glucosamine sulphate have beenadvocated as safe and effective options for the treatment ofosteoarthritis, a recent clinical trial revealed that both treatmentsdid not reduce pain associated to osteoarthritis. (Clegg et al., 2006).

Stimulation of the anabolic processes, blocking catabolic processes, ora combination of these two, may result in stabilization of thecartilage, and perhaps even reversion of the damage, and thereforeprevent further progression of the disease. Therapeutic methods for thecorrection of the articular cartilage lesions that appear during theosteoarthritic disease have been developed, but so far none of them havebeen able to mediate the regeneration of articular cartilage in situ andin vivo. Taken together, no disease modifying osteoarthritic drugs areavailable.

Vandeghinste et al. (WO 2005/124342) discovered JAK1 as a target whoseinhibition might have therapeutic relevance for several diseasesincluding OA. Knockout of the JAK1 gene in mice demonstrated that JAK1plays essential and non-redundant roles during development: JAK1−/− micedied within 24 h after birth and lymphocyte development was severelyimpaired. Moreover, JAK1−/− cells were not, or less, reactive tocytokines that use class II cytokine receptors, cytokine receptors thatuse the gamma-c subunit for signaling and the family of cytokinereceptors that use the gp130 subunit for signaling (Rodig et al., 1998).

Various groups have implicated JAK-STAT signaling in chondrocytebiology. Li et al. (2001) showed that Oncostatin M induces MMP and TIMP3gene expression in primary chondrocytes by activation of JAK/STAT andMAPK signaling pathways. Osaki et al. (2003) showed thatinterferon-gamma mediated inhibition of collagen II in chondrocytesinvolves JAK-STAT signaling. IL1-beta induces cartilage catabolism byreducing the expression of matrix components, and by inducing theexpression of collagenases and inducible nitric oxide synthase (NOS2),which mediates the production of nitric oxide (NO). Otero et al., (2005)showed that leptin and IL1-beta synergistically induced NO production orexpression of NOS2 mRNA in chondrocytes, and that that was blocked by aJAK inhibitor. Legendre et al. (2003) showed that IL6/IL6 Receptorinduced downregulation of cartilage-specific matrix genes collagen II,aggrecan core and link protein in bovine articular chondrocytes, andthat this was mediated by JAK/STAT signaling. Therefore, theseobservations suggest a role for JAK kinase activity in cartilagehomeostasis and therapeutic opportunities for JAK kinase inhibitors.

JAK family members have been implicated in additional conditionsincluding myeloproliferative disorders (O'Sullivan et al, 2007, MolImmunol 44(10):2497-506), where mutations in JAK2 have been identified.This indicates that inhibitors of JAK in particular JAK2 may also be ofuse in the treatment of myeloproliferative disorders. Additionally, theJAK family, in particular JAK1, JAK2 and JAK3, has been linked tocancers, in particular leukaemias e.g. acute myeloid leukaemia(O'Sullivan et al, 2007, Mol Immunol 44(10):2497-506; Xiang et al.,2008, “Identification of somatic JAK1 mutations in patients with acutemyeloid leukemia” Blood First Edition Paper, prepublished online Dec.26, 2007; DOI 10.1182/blood-2007-05-090308) and acute lymphoblasticleukaemia (Mullighan et al, 2009) or solid tumours e.g. uterineleiomyosarcoma (Constantinescu et al., 2007, Trends in BiochemicalSciences 33(3): 122-131), prostate cancer (Tam et al., 2007, BritishJournal of Cancer, 97, 378-383). These results indicate that inhibitorsof JAK, in particular of JAK1 and/or JAK2, may also have utility in thetreatment of cancers (leukaemias and solid tumours e.g. uterineleiomyosarcoma, prostate cancer).

In addition, Castleman's disease, multiple myeloma, mesangialproliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma arelikely due to hypersecretion of the cytokine IL-6, whose biologicaleffects are mediated by intracellular JAK-STAT signaling (Tetsuji Naka,Norihiro Nishimoto and Tadamitsu Kishimoto, Arthritis Res 2002, 4 (suppl3):S233-S242). This result shows that inhibitors of JAK, may also findutility in the treatment of said diseases.

The current therapies are not satisfactory and therefore there remains aneed to identify further compounds that may be of use in the treatmentof inflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, e.g.osteoarthritis, and rheumatoid arthritis, in particular rheumatoidarthritis. The present invention therefore provides a compound, methodsfor its manufacture and pharmaceutical compositions comprising thecompound of the invention together with a suitable pharmaceuticalcarrier. The present invention also provides for the use of the compoundof the invention in the preparation of a medicament for the treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, e.g.osteoarthritis, and rheumatoid arthritis, in particular rheumatoidarthritis.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the compound of theinvention is able to act as inhibitor of JAK and that it is useful forthe treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons. In a specific aspect the compound of the invention is aninhibitor of JAK1 and/or JAK2. The present invention also providesmethods for the production of this compound, pharmaceutical compositionscomprising this compound and methods for treating inflammatoryconditions, autoimmune diseases, proliferative diseases, allergy,transplant rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of TL6 or interferons, by administering the compoundof the invention.

Accordingly, in a first aspect of the invention, the compound of theinvention provided is according to Formula (I):

In a particular embodiment the compound of the invention is an inhibitorof JAK1 and/or JAK2.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising the compound of the invention, and apharmaceutical carrier, excipient or diluent. Moreover, the compound ofthe invention, useful in the pharmaceutical compositions and treatmentmethods disclosed herein, is pharmaceutically acceptable as prepared andused. In this aspect of the invention, the pharmaceutical compositionmay additionally comprise further active ingredients suitable for use incombination with the compound of the invention.

In a further aspect of the invention, this invention provides a methodof treatment or prophylaxis of a mammal susceptible to or afflicted witha condition from among those listed herein, and particularly,inflammatory conditions, autoimmunc diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and diseasesassociated with hypersecretion of IL6 or interferons, which methodcomprises administering an effective amount of the pharmaceuticalcomposition or compound of the invention as described herein. In aparticular embodiment the condition is associated with aberrant JAKactivity, and more particularly JAK1 and/or JAK2 activity.

In a further aspect, the invention provides the compound of theinvention or a pharmaceutical composition comprising the compound of theinvention for use as a medicament. In a specific embodiment, saidpharmaceutical composition additionally comprises a further activeingredient.

In a further aspect, the present invention provides the compound of theinvention for use in the treatment or prophyaxis of a condition selectedfrom those listed herein, particularly inflammatory conditions,autoimmune diseases, proliferative diseases, allergy, transplantrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons.

In yet another method of treatment aspect, this invention provides amethod of treatment or prophylaxis of a mammal susceptible to orafflicted with a selected from those listed herein, particularlyinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, and comprisesadministering an effective amount of the pharmaceutical composition orthe compound of the invention described herein for the treatment orprophylaxis of said condition. In a specific aspect the condition iscausally related to abnormal JAK activity and more particularly JAK1and/or JAK2 activity.

In a further aspect, the present invention provides the compound of theinvention for use in the treatment or prophylaxis of a conditionselected from those listed herein, particularly inflammatory conditions,autoimmune diseases, proliferative diseases, allergy, transplantrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons.

In additional aspects, this invention provides methods for synthesizingthe compound of the invention, with representative synthetic protocolsand pathways disclosed later on herein.

Accordingly, it is a principal object of this invention to provide anovel compound, which can modify the activity of JAK and thus prevent ortreat any conditions that may be causally related thereto. In a specificaspect the compound of the invention can modulate the activity of JAK1and/or JAK2.

It is further an object of this invention to provide a compound that cantreat or alleviate conditions or symptoms of same, such as inflammatoryconditions, autoimmune diseases, proliferative diseases, allergy,transplant rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or interferons.

A still further object of this invention is to provide a pharmaceuticalcomposition that may be used in the treatment or prophylaxis of avariety of disease states, such as inflammatory conditions, autoimmuncdiseases, proliferative diseases, allergy, transplant rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or interferons. In a specific embodiment the disease isassociated with JAK1 and/or JAK2 activity, in particular inflammatoryconditions, autoimmune diseases, proliferative diseases, allergy,transplant rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or interferons.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the term “substituted” is to be defined as set out below. It should befurther understood that the terms “groups” and “radicals” can beconsidered interchangeable when used herein.

The articles “a” and “an” may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein the term ‘JAK’ relates to the family of Janus kinases(JAKs) which are cytoplasmic tyrosine kinases that transduce cytokinesignaling from membrane receptors to STAT transcription factors. FourJAK family members are described, JAK1, JAK2, JAK3 and TYK2 and the termJAK may refer to all the JAK family members collectively or one or moreof the JAK family members as the context indicates.

‘Alkoxy’ refers to the group —OR²⁶ where R²⁶ is C₁-₈ alkyl. Particularalkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6carbon atoms. Further particular alkoxy groups have between 1 and 4carbon atoms.

‘Alkyl’ means straight or branched aliphatic hydrocarbon having 1 to 20carbon atoms. Particular alkyl has 1 to 8 carbon atoms. More particularis lower alkyl which has 1 to 6 carbon atoms. A further particular grouphas 1 to 4 carbon atoms. Exemplary straight chained groups includemethyl, ethyl n-propyl, and n-butyl. Branched means that one or morelower alkyl groups such as methyl, ethyl, propyl or butyl is attached toa linear alkyl chain, exemplary branched chain groups include isopropyl,iso-butyl, t-butyl and isoamyl.

‘Aryl’ refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. In particular aryl refers to an aromatic ringstructure, monocyclic or polyyclic, with the number of ring atomsspecified. Specifically, the term includes groups that include from 6 to10 ring members. Where the aryl group is a monocyclic ring system itpreferentially contains 6 carbon atoms. Particularly aryl groups includephenyl, naphthyl, indenyl, and tetrahydronaphthyl.

‘Substituted’ refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable salt’ refers to a salt of the compound ofthe invention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g. an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term‘pharmaceutically acceptable cation’ refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which the compound of the invention isadministered.

‘Prodrugs’ refers to compounds, including derivatives of the compound ofthe invcntion,which have cleavable groups and become by solvolysis orunder physiological conditions the compound of the invention which arepharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding Conventional solvents include water, ethanol,acetic acid and the like. The compound of the invention may be preparede.g. in crystalline form and may be solvated or hydrated. Suitablesolvates include pharmaceutically acceptable solvates, such as hydrates,and further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. ‘Solvate’ encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

‘Subject’ includes humans The terms ‘human’, ‘patient’ and ‘subject’ areused interchangeably herein.

‘Therapeutically effective amount’ means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringor developing a disease or disorder (i.e. causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e. arresting thedisease or reducing the manifestation, extent or severity of at leastone of the clinical symptoms thereof). In another embodiment ‘treating’or ‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. Tn yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or disorder,either physically, (e.g. stabilization of a discernible symptom),physiologically, (e.g. stabilization of a physical parameter), or both.In a further embodiment, “treating” or “treatment” relates to slowingthe progression of the disease.

As used herein the term ‘allergy’ refers to the group of conditionscharacterized by a hypersensitivity disorder of the immune systemincluding, allergic airway disease (e.g. asthma, rhinitis), sinusitis,eczema and hives, as well as food allergies or allergies to insectvenom.

As used herein the term ‘inflammatory condition(s)’ refers to the groupof conditions including, rheumatoid arthritis, osteoarthritis, juvenileidiopathic arthritis, psoriasis, psoriatic arthritis, allergic airwaydisease (e.g. asthma, rhinitis), inflammatory bowel diseases (e.g.Crohn's disease, ulcerative colitis), endotoxin-driven disease states(e.g. complications after bypass surgery or chronic endotoxin statescontributing to e.g. chronic cardiac failure), and related diseasesinvolving cartilage, such as that of the joints. Partcicularly the termrefers to rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma) and inflammatory bowel diseases.

As used herein the term ‘autoimmune disease(s)’ refers to the group ofdiseases including obstructive airways disease, including conditionssuch as COPD, asthma (e.g intrinsic asthma, extrinsic asthma, dustasthma, infantily asthma) particularly chronic or inveterate asthma (forexample late asthma and airway hyperreponsiveness), bronchitis,including bronchial asthma, systemic lupus erythematosus (SLE),cutaneous lupus erythrematosis, lupus nephritis, dermatomyositis,Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, typeI diabetes mellitus and complications associated therewith, atopiceczema (atopic dermatitis), contact dermatitis and further eczematousdermatitis, inflammatory bowel disease (e.g. Crohn's disease andulcerative colitis), psoriatic arthritis, ankylosing spondylitis,juvenile ideopathic arthritis, atherosclerosis and amyotrophic lateralsclerosis. Particularly the term refers to COPD, asthma, systemic lupuserythematosis, type I diabetes mellitus and inflammatory bowel disease.

As used herein the term ‘proliferative disease(s)’ refers to conditionssuch as cancer (e.g. uterine leiomyosarcoma or prostate cancer),myeloproliferative disorders (e.g. polycythemia vera, essentialthrombocytosis and myelofibrosis), leukemia (e.g. acute myeloidleukaemia, acute and chronic lymphoblastic leukemia), multiple myeloma,psoriasis, restenosis, scleroderma or fibrosis. In particular the termrefers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term ‘cancer’ refers to a malignant or benign growthof cells in skin or in body organs, for example but without limitation,breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancertends to infiltrate into adjacent tissue and spread (metastasise) todistant organs, for example to bone, liver, lung or the brain. As usedherein the term cancer includes both metastatic rumour cell types, suchas but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such asbut not limited to, colorectal cancer, prostate cancer, small cell lungcancer and non-small cell lung cancer, breast cancer, pancreatic cancer,bladder cancer, renal cancer, gastric cancer, glioblastoma, primaryliver cancer, ovarian cancer, prostate cancer and uterineleiomyosarcoma.

As used herein the term ‘leukemia’ refers to neoplastic diseases of theblood and blood forming organs. Such diseases can cause bone marrow andimmune system dysfunction, which renders the host highly susceptible toinfection and bleeding. In particular the term leukemia refers to acutemyeloid leukaemia (AML) and acute lymphoblastic leukemia (ALL) andchronic lymphoblastic leukaemia (CLL).

As used herein the term ‘transplant rejection’ refers to the acute orchronic rejection of cells, tissue or solid organ allo- or xenografts ofe.g. pancreatic islets, stem cells, bone marrow, skin, muscle, cornealtissue, neuronal tissue, heart, lung, combined heart-lung, kidney,liver, bowel, pancreas, trachea or oesophagus, or graft-versus-hostdiseases.

As used herein the term ‘diseases involving impairment of cartilageturnover’ includes conditions such as osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,endemic forms of arthritis like osteoarthritis deformans endemica,Mseleni disease and Handigodu disease; degeneration resulting fromfibromyalgia, systemic lupus erythematosus, scleroderma and ankylosingspondylitis.

As used herein the term ‘congenital cartilage malformation(s)’ includesconditions such as hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

As used herein the term ‘disease(s) associated with hypersecretion ofIL6’ includes conditions such as Castleman's disease, multiple myeloma,psoriasis, Kaposi's sarcoma and/or mesangial proliferativeglomerulonephritis.

As used herein the term ‘disease(s) associated with hypersecretion ofinterferons includes conditions such as systemic and cutaneous lupuserythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome,psoriasis, rheumatoid arthritis.

‘Compound of the invention’, and equivalent expressions, are meant toembrace the compound of the Formula as herein described, whichexpression includes the biologically active metabolites,pharmaceutically acceptable salts, and the solvates, e.g. hydrates, andthe solvates of the pharmaceutically acceptable salts where the contextso permits. Similarly, reference to intermediates, whether or not theythemselves are claimed, is meant to embrace their salts, and solvates,where the context so permits.

When ranges are referred to herein, for example but without limitation,C₁₋₈ alkyl, the citation of a range should be considered arepresentation of each member of said range.

Other derivatives of the compound of this invention have activity inboth their acid and acid derivative forms, but in the acid sensitiveform often offers advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare particularly useful prodrugs. In some cases it is desirable toprepare double ester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particular such prodrugs are the C₁ toC₈ alkyl, C₂-₈ alkenyl, aryl, C₇-₁₂ substituted aryl, and C₇-₁₂arylalkyl esters of the compound of the invention.

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Further, compounds may beprepared that are substituted with positron emitting isotopes, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy.

All isotopic variants of the compounds provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed ‘isomers’. Isomersthat differ in the arrangement of their atoms in space are termed‘stereoisomers’.

Stereoisomers that are not mirror images of one another are termed‘diastereomers’ and those that are non-superimposable mirror images ofeach other are termed ‘enantiomers’. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e. as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a ‘racemic mixture’.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

The Compound

The present invention is based on the identification that the compoundof the invention is an inhibitor of JAK and that it may be useful forthe treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons. The present invention also provides methods for theproduction of the compound of the invention, pharmaceutical compositionscomprising the compound of the invention and methods for treatinginflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, by administeringthe compound of the invention. In a specific embodiment the compound ofthe invention is an inhibitor of JAK1 and/or JAK2.

Accordingly, in a first aspect of the invention, the compound of theinvention is according to Formula (I):

In one embodiment the compound of the invention is not an isotopicvariant.

In one aspect the compound of the invention is present as the free base.

In one aspect the compound of the invention is a pharmaceuticallyacceptable salt.

In one aspect the compound of the invention is a solvate of thecompound.

In one aspect the compound of the invention is a solvate of apharmaceutically acceptable salt of a compound.

In certain aspects, the present invention provides prodrugs andderivatives of the compound of the invention. Prodrugs arc derivativesof the compound of the invention, which have metabolically cleavablegroups and become by solvolysis or under physiological conditions thecompound of the invention, which are pharmaceutically active, in vivo.Such examples include, but are not limited to, choline ester derivativesand the like, N-alkylmorpholine esters and the like.

Other derivatives of the compound of this invention have activity inboth their acid and acid derivative forms, but the acid sensitive formoften offers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare preferred prodrugs. In some cases it is desirable to prepare doubleester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the C₁ to C₈alkyl, C₂-₈ alkenyl, aryl, C₇-₁₂ substituted aryl, and C₇-₁₂ arylalkylesters of the compound of the invention.

The compound of the invention is a novel inhibitor of JAK. Inparticular, the compound is a potent inhibitor of JAK1 and/or JAK2,however it may inhibit TYK2 and JAK3 with a lower potency.

Pharmaceutical Compositions

When employed as a pharmaceutical, the compound of the invention istypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound. Generally,the compound of this invention is administered in a pharmaceuticallyeffective amount. The amount of the compound actually administered willtypically be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual compound administered, the age, weight,and response of the individual patient, the severity of the patient'ssymptoms, and the like.

The pharmaceutical compositions of the invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intra-articular, intravenous, intramuscular, and intranasal. Dependingon the intended route of delivery, a compound of this invention ispreferably formulated as either injectable or oral compositions or assalves, as lotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term ‘unit dosage forms’ refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as a ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

The compound of the invention can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, MackPublishing Company, Easton, Pennsylvania, which is incorporated hereinby reference.

The compound of the invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1—Tablets

The compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate may be added as a lubricant. The mixture may beformed into 240-270 mg tablets (80-90 mg of active amide compound pertablet) in a tablet press.

Formulation 2—Capsules

The compound of the invention may be admixed as a dry powder with astarch diluent in an approximate 1:1 weight ratio. The mixture may befilled into 250 mg capsules (125 mg of active amide compound percapsule).

Formulation 3—Liquid

The compound of the invention (125 mg), may be admixed with sucrose(1.75 g) and xanthan gum (4 mg) and the resultant mixture may beblended, passed through a No. 10 mesh U.S. sieve, and then mixed with apreviously made solution of microcrystalline cellulose and sodiumcarboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10mg), flavor, and color may be diluted with water and added withstirring. Sufficient water may then be added with stirring. Furthersufficient water may be then added to produce a total volume of 5 mL.

Formulation 4—Tablets

The compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate may be added as a lubricant. The mixture may beformed into 450-900 mg tablets (150-300 mg of active amide compound) ina tablet press.

Formulation 5—Injection

The compound of the invention may be dissolved or suspended in abuffered sterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

Formulation 6—Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted atabout 75° C. and then a mixture of the compound of the invention (50 g)methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate(10 g), and propylene glycol (120 g) dissolved in water (about 370 g)may be added and the resulting mixture may be stirred until it congeals.

Methods of Treatment

The compound of the invention may be used as a therapeutic agent for thetreatment or prophylaxis of conditions in mammals that are causallyrelated or attributable to aberrant activity of JAK. In particular,conditions related to aberrant activity of JAK1 and/or JAK2.

Accordingly, the compounds and pharmaceutical compositions of theinvention find use as therapeutics for the treatment or prophylaxis ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and diseasesassociated with hypersecretion of IL6 or interferons, in mammalsincluding humans.

In one aspect, the present invention provides the compound of theinvention, or a pharmaceutical composition comprising the compound ofthe invention for use as a medicament.

In a method of treatment aspects, this invention provides methods oftreatment or prophylaxis of a mammal susceptible to or afflicted with anallergic reaction. In a specific embodiment, the invention providesmethods of treatment or prophylaxis of allergic airway disease,sinusitis, eczema and hives, food allergies or allergies to insectvenom.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of an allergicreaction. In a specific embodiment, the invention provides methods oftreatment or prophylaxis of allergic airway disease, sinusitis, eczemaand hives, food allergies or allergies to insect venom.

In additional method of treatment aspects, this invention providesmethods of treatment or prophylaxis of a mammal susceptible to orafflicted with an inflammatory condition. The methods compriseadministering an effective amount of one or more of the pharmaceuticalcompositions or compound of the invention herein described for thetreatment or prophylaxis of said condition. In a specific embodiment,the inflammatory condition is selected from rheumatoid arthritis,osteoarthritis, allergic airway disease (e.g. asthma) and inflammatorybowel diseases.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of an inflammatorycondition. In a specific embodiment, the inflammatory condition isselected from rheumatoid arthritis, osteoarthritis, allergic airwaydisease (e.g. asthma) and inflammatory bowel diseases.

In additional method of treatment aspects, this invention providesmethods of treatment or prophylaxis of a mammal susceptible to orafflicted with an autoimmune disease. The methods comprise administeringan effective amount of one or more of the pharmaceutical compositions orcompound of the invention herein described for the treatment orprophylaxis of said condition. In a specific embodiment, the autoimmunedisease is selected from COPD, asthma, systemic lupus erythematosis,type I diabetes mellitus, psoriatic arthritis, ankylosing spondylitis,juvenile ideopathic arthritis, and inflammatory bowel disease.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of an autoimmunedisease. In a specific embodiment, the autoimmune disease is selectedfrom COPD, asthma, systemic lupus erythematosis, type I diabetesmellitus, psoriatic arthritis, ankylosing spondylitis, juvenileideopathic arthritis, and inflammatory bowel disease. In a more specificembodiment, the autoimmune disease is systemic lupus erythematosis.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted witha proliferative disease, in particular cancer (e.g. solid tumors such asuterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL orCLL), multiple myeloma and/or psoriasis.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of a proliferativedisease, in particular cancer (e.g. solid tumors such as uterineleiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL),multiple myeloma and/or psoriasis.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withtransplant rejection. In a specific embodiment, the invention providesmethods of treatment or prophylaxis of organ transplant rejection.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of transplantrejection. In a specific embodiment, the invention provides methods oftreatment or prophylaxis of organ transplant rejection.

In a method of treatment aspect, this invention provides a method oftreatment or prophylaxis in a mammal susceptible to or afflicted withdiseases involving impairment of cartilage turnover, which methodcomprises administering a therapeutically effective amount of thecompound of the invention, or one or more of the pharmaceuticalcompositions herein described.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of diseases involvingimpairment of cartilage turnover.

The present invention also provides a method of treatment or prophylaxisof congenital cartilage malformations, which method comprisesadministering an effective amount of one or more of the pharmaceuticalcompositions or compound of the invention herein described.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of congenitalcartilage malformations.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withdiseases associated with hypersecretion of IL6, in particularCastleman's disease or mesangial proliferative glomerulonephritis.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of diseases associatedwith hypersecretion of IL6, in particular Castleman's disease ormesangial proliferative glomerulonephritis.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withdiseases associated with hypersecretion of IL6, in particular systemicand cutaneous lupus erythematosis, lupus nephritis, dermatomyositis,Sjogren's syndrome, psoriasis, rheumatoid arthritis.

In another aspect the present invention provides the compound of theinvention for use in the treatment or prophylaxis of diseases associatedwith hypersecretion of IL6, in particular systemic and cutaneous lupuserythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome,psoriasis, and rheumatoid arthritis.

As a further aspect of the invention there is provided the compound ofthe invention for use as a pharmaceutical especially in the treatment orprophylaxis of the aforementioned conditions and diseases. Also providedherein is the use of the present compounds in the manufacture of amedicament for the treatment or prophylaxis of one of the aforementionedconditions and diseases.

A particular regimen of the present method comprises the administrationto a subject suffering from a disease involving inflammation, inparticular rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma) and inflammatory bowel diseases, of an effective amount ofthe compound of the invention for a period of time sufficient to reducethe level of inflammation in the subject, and preferably terminate theprocesses responsible for said inflammation. A special embodiment of themethod comprises administering an effective amount of the compound ofthe invention to a subject patient suffering from or susceptible to thedevelopment of rheumatoid arthritis, for a period of time sufficient toreduce or prevent, respectively, inflammation in the joints of saidpatient, and preferably to terminate, the processes responsible for saidinflammation.

A further particular regimen of the present method comprises theadministration to a subject suffering from a disease conditioncharacterized by cartilage or joint degradation (e.g. rheumatoidarthritis and/or osteoarthritis) of an effective amount of the compoundof the invention for a period of time sufficient to reduce andpreferably to terminate the self-perpetuating processes responsible forsaid degradation. A particular embodiment of the method comprisesadministering of an effective amount of the compound of the invention toa subject patient suffering from or susceptible to the development ofosteoarthritis, for a period of time sufficient to reduce or prevent,respectively, cartilage degradation in the joints of said patient, andpreferably to terminate, the self-perpetuating processes responsible forsaid degradation. In a particular embodiment said compound may exhibitcartilage anabolic and/or anti-catabolic properties.

Injection dose levels range from about 0.1 mg/kg/h to at least 10mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h.A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more mayalso be administered to achieve adequate steady state levels. Themaximum total dose is not expected to exceed about 2 g/day for a 40 to80 kg human patient.

For the prophylaxis and/or treatment of long-term conditions, such asdegenerative conditions, the regimen for treatment usually stretchesover many months or years, so oral dosing is preferred for patientconvenience and tolerance. With oral dosing, one to five and especiallytwo to four and typically three oral doses per day are representativeregimens. Using these dosing patterns, each dose provides from about0.01 to about 20 mg/kg of the compound of the invention, with particulardoses each providing from about 0.1 to about 10 mg/kg and especiallyabout 1 to about 5 mg/kg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of a condition, the compound of theinvention will be administered to a patient at risk for developing thecondition, typically on the advice and under the supervision of aphysician, at the dosage levels described above. Patients at risk fordeveloping a particular condition generally include those that have afamily history of the condition, or those who have been identified bygenetic testing or screening to be particularly susceptible todeveloping the condition.

The compound of the invention can be administered as the sole activeagent or it can be administered in combination with other therapeuticagents, including other compounds that demonstrate the same or a similartherapeutic activity and that are determined to safe and efficacious forsuch combined administration. In a specific embodiment,co-administration of two (or more) agents allows for significantly lowerdoses of each to be used, thereby reducing the side effects seen.

In one embodiment, the compound of the invention or a pharmaceuticalcomposition comprising the compound of the invention is administered asa medicament. In a specific embodiment, said pharmaceutical compositionadditionally comprises a further active ingredient.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of adisease involving inflammation, particular agents include, but are notlimited to, immunoregulatory agents e.g. azathioprine, corticosteroids(e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A,tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OKT3, e.g.Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, andpiroxicam.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofarthritis (e.g. rheumatoid arthritis), particular agents include but arenot limited to analgesics, non-steroidal anti-inflammatory drugs(NSAIDS), steroids, synthetic DMARDS (for example but without limitationmethotrexate, leflunomide, sulfasalazine, auranofin, sodiumaurothiomalate, penicillamine, chloroquine, hydroxychloroquine,azathioprine, Tofacitinib, Fostamatinib, and cyclosporin), andbiological DMARDS (for example but without limitation Infliximab,Etanercept, Adalimumab, Rituximab, and Abatacept).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofproliferative disorders, particular agents include but are not limitedto: methotrexate, leukovorin, adriamycin, prenisone, bleomycin,cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrolacetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors(e.g. Iressa®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™),proteasome inhibitors (e.g. Velcade™), Glivec® and hsp90 inhibitors(e.g. 17-AAG). Additionally, the compound of the invention may beadministered in combination with other therapies including, but notlimited to, radiotherapy or surgery. In a specific embodiment theproliferative disorder is selected from cancer, myeloproliferativedisease or leukaemia.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofautoimmune diseases, particular agents include but are not limited to:glucocorticoids, cytostatic agents (e.g. purine analogs), alkylatingagents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas,platinum compounds, and others), antimetabolites (e.g. methotrexate,azathioprine and mercaptopurine), cytotoxic antibiotics (e.g.dactinomycin anthracyclines, mitomycin C, bleomycin, and mithramycin),antibodies (e.g. anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonalantibodies, Atgam® and Thymoglobulinc®), cyclosporin, tacrolimus,rapamycin (sirolimus), interferons (e.g. IFN-β), TNF binding proteins(e.g. infliximab (Remicade™) etanercept (Enbrel™), or adalimumab(Humira™)), mycophenolate, Fingolimod and Myriocin.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis oftransplant rejection, particular agents include but are not limited to:calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)), mTORinhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g.azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone,hydrocortisone), Antibodies (e.g. monoclonal anti-IL-2Rα receptorantibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies(e.g. anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of asthmaand/or rhinitis and/or COPD, particular agents include but are notlimited to: beta₂-adrenoceptor agonists (e.g. salbutamol, levalbuterol,terbutaline and bitolterol), epinephrine (inhaled or tablets),anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral orinhaled) Long-acting β₂-agonists (e.g. salmeterol, formoterol,bambuterol, and sustained-release oral albuterol), combinations ofinhaled steroids and long-acting bronchodilators (e.g.fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonistsand synthesis inhibitors (e.g. montelukast, zafirlukast and zileuton),inhibitors of mediator release (e.g. cromoglycate and ketotifen),biological regulators of IgE response (e.g. omalizumab), antihistamines(e.g. ceterizine, cinnarizine, fexofenadine) and vasoconstrictors (e.g.oxymethazoline, xylomethazoline, nafazoline and tramazoline).

Additionally, the compound of the invention may be administered incombination with emergency therapies for asthma and/or COPD. Suchtherapies include oxygen or heliox administration, nebulized salbutamolor terbutaline (optionally combined with an anticholinergic (e.g.ipratropium), systemic steroids (oral or intravenous, e.g. prednisone,prednisolone, methylprednisolone, dexamethasone, or hydrocortisone),intravenous salbutamol, non-specific beta-agonists, injected or inhaled(e.g. epinephrine, isoetharine, isoproterenol, metaproterenol),anticholinergics (IV or nebulized, e.g. glycopyrrolate, atropine,ipratropium), methylxanthines (theophylline, aminophylline,bamiphylline), inhalation anesthetics that have a bronchodilatory effect(e.g. isoflurane, halothane, enflurane), ketamine and intravenousmagnesium sulfate.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofinflammatory bowel disease (IBD), particular agents include but are notlimited to: glucocorticoids (e.g. prednisone, budesonide) syntheticdisease modifying, immunomodulatory agents (e.g. methotrexate,leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurineand cyclosporin) and biological disease modifying, immunomodulatoryagents (infliximab, adalimumab, rituximab, and abatacept).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of SLE,particular agents include but are not limited to: Disease-modifyingantirheumatic drugs (DMARDs) such as antimalarials (e.g. plaqucnil,hydroxychloroquine), immunosuppressants (e.g. methotrexate andazathioprine), cyclophosphamide and mycophenolic acid; immunosuppressivedrugs and analgesics, such as nonsteroidal anti-inflammatory drugs,opiates (e.g. dextropropoxyphene and co-codamol), opioids (e.g.hydrocodone, oxycodone, MS Contin, or methadone) and the fentanylduragesic transdermal patch.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofpsoriasis, particular agents include but are not limited to: topicaltreatments such as bath solutions, moisturizers, medicated creams andointments containing coal tar, dithranol (anthralin), corticosteroidslike desoximetasone (Topicort™), fluocinonide, vitamin D₃ analogues (forexample, calcipotriol), Argan oiland retinoids (etretinate, acitretin,tazarotene), systemic treatments such as methotrexate, cyclosporine,retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolatemofetil, azathioprine, tacrolimus, fumaric acid esters or biologics suchas Amevive™, Enbrel™, Humira™, Remicade™, Raptiva™ and ustekinumab (aIL-12 and IL-23 blocker). Additionally, the compound of the inventionmay be administered in combination with other therapies including, butnot limited to phototherapy, or photochemotherapy (e.g. psoralen andultraviolet A phototherapy (PUVA)).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofallergic reaction, particular agents include but are not limited to:antihistamines (e.g. cetirizine, diphenhydramine, fexofenadine,levocetirizine), glucocorticoids (e.g. prednisone, betamethasone,beclomethasone, dexamethasone), epinephrine, theophylline oranti-leukotrienes (e.g. montelukast or zafirlukast), anti-cholinergicsand decongestants.

By co-administration is included any means of delivering two or moretherapeutic agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation, i.e. as asingle pharmaceutical composition, this is not essential. The agents maybe administered in different formulations and at different times.

General Synthetic Procedures General

The compound of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e. reaction temperatures, times, mole ratios of reactants,solvents, pressures, etc.) are given, other process conditions can alsobe used unless otherwise stated. Optimum reaction conditions may varywith the particular reactants or solvent used, but such conditions canbe determined by one skilled in the art by routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991, andreferences cited therein.

The following methods are presented with details as to the preparationof the compound of the invention as defined hereinabove and thecomparative examples. The compound of the invention may be prepared fromknown or commercially available starting materials and reagents by oneskilled in the art of organic synthesis.

All reagents were of commercial grade and were used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents were used for reactions conducted under inertatmosphere. Reagent grade solvents were used in all other cases, unlessotherwise specified. Column chromatography was performed on silica gel60 (35-70 μm). Thin layer chromatography was carried out usingpre-coated silica gel F-254 plates (thickness 0.25 mm) ¹H NMR spectrawere recorded on a Bruker DPX 400 NMR spectrometer (400 MHz). Chemicalshifts (δ) for ¹H NMR spectra are reported in parts per million (ppm)relative to tetramethylsilane (δ 0.00) or the appropriate residualsolvent peak, i.e. CHCl₃ (δ 7.27), as internal reference. Multiplicitiesare given as singlet (s), doublet (d), triplet (t), quartet (q),multiplet (m) and broad (br). Coupling constants (J) are given in Hz.Electrospray MS spectra were obtained on a Micromass platform LC/MSspectrometer. Columns Used for LCMS analysis: Hichrom, KromasilEternity, 2.5 μm C18, 150×4.6 mm, Waters Xbridge 5 μm C18 (2), 250×4.6mm (ref 86003117), Waters Xterra MS 5 μm C18, 100×4.6 mm (Plus guardcartridge) (ref 186000486), Gemini-NX 3 μm C18 100×3.0 mm (ref00D-4453-Y0), Phenomenex Luna 5 μm C18 (2), 100×4.6 mm (Plus guardcartridge) (ref 00D-4252-E0), Kinetix fused core 2.7 μm C18 100×4.6 mm(ref 00D-4462-E0), Supelco, Ascentis® Express C18 (ref 53829-U), orHichrom Halo C18, 2.7 μm C18, 150×4.6 mm (ref 92814-702). LC-MS wererecorded on a Waters Micromass ZQ coupled to a HPLC Waters 2795,equipped with a UV detector Waters 2996. LC were also run on a HPLCAgilent 1100 coupled to a UV detector Agilent G1315A. Preparative HPLC:Waters XBridge Prep C18 5 μm ODB 19 mm ID×100 mm L (Part No. 186002978).All the methods are using MeCN/H₂O gradients. H₂O contains either 0.1%TFA or 0.1% NH₃.

List of abbreviations used in the experimental section:

DCM Dichloromethane TFA Trifluoroacetic acid DiPEAN,N-diisopropylethylamine THF Tetrahydrofuran MeCN Acetonitrile NMRNuclear Magnetic Resonnance BOC tert-Butyloxy-carbonyl DMSODimethylsulfoxide DMF N,N-dimethylformamide LC-MS Liquid Chromatography-Mass Cat. Catalytic amount Spectrometry Ppm part-per-million PBSTPhosphate buffered saline with Pd/C Palladium on Charcoal 10% Tween 3.2mM Na₂HPO₄, 0.5 PMB Para-methoxy-benzyl mM KH₂PO₄, 1.3 mM KCl, 135 PyBOPbenzotriazol-1-yl-oxy-tris- mM NaCl, 0.05% Tween 20,pyrrolidino-phosphonium pH 7.4 hexafluoroborate APMA4-aminophenylmercuric acetate EtOAc ethyl acetate DMEM Dulbecco'sModified Eagle APCI atmospheric pressure Medium chemical ionization FBSFetal bovine serum Rt retention time hCAR human cellular adenovirus ssinglet receptor br s broad singlet 3-MOI multiplicity of infection of 3m multiplet dNTP deoxyribonucleoside min minute triphosphate mLmilliliter QPCR quantitative polymerase chain μL microliter reaction ggram cDNA copy deoxyribonucleic acid mg milligram GAPDH Glyceraldehydephosphate dehydrogenase PdCl₂dppf [1,1′- h hourBis(diphenylphosphino)ferro- mmol millimoles cene] dichloropalladium(II)HATU O-(Benzotriazol-1-yl)- TEA TriethylamineN,N,N′,N′-tetramethyluronium MMP Matrix Metallo Proteinasehexafluorophosphate NHAC Normal Human Articular HPLC High pressureliquid Chondrocytes chromatography shRNA short hairpin RNA PS- Polymersupported- RNA Ribonucleic acid NMe₃BH₃CN NMe₃BH₃CN Ad-Si RNA Adenoviralencoded siRNA NMP N-Methylpyrrolidone

Synthetic Preparation of the Compound of the Invention

The compound of the invention can be produced according to the followingscheme.

Compound 1:N-4-(3-fluoro-4-((4-(methylsulfonyl)piperazin-1-yl)methyl)phenyl)pyrazolo[1,5-a]pyridin-2-yl)acetamide

Step i): 3-bromo-2-cyanomethylpyridine (Intermediate 1)

Dry THF (100 mL) was cooled to −78° C. and n-butyllithium (2.5 M inhexanes; 23 mL, 58 mmol) was added. Acetonitrile (3.3 mL, 64 mmol) wasadded dropwise maintaining the temperature below −60° C. A whiteprecipitate formed and the reaction mixture was stirred at −78° C. for45 min. A solution of 2,3-dibromopyridine (2.0 g, 8.4 mmol) in dry THF(10 mL) was added dropwise and the reaction mixture stirred at −78° C.for 1.5 h then allowed to warm to room temperature. The reaction wasquenched by the dropwise addition of water. The aqueous was extractedwith DCM (×3) and the combined organics washed with brine, dried (MgSO₄)and concentrated in vacuo. (3-Bromo-pyridin-2-yl)-acetonitrile(Intermediate 1) was purified by flash column chromatography.

Step ii) 2-(3-Bromo-pyridin-2-yl)-N-hydroxy-acetamidine (Intermediate 2)

A vigorously stirred mixture of hydroxylamine hydrochloride (924 mg,13.3 mmol), water (4 mL), NaHCO₃ (1.12 g, 13.3 mmol),3-bromo-2-cyanomethylpyridine (Intermediate 1) (1.31 g, 6.65 mmol) andethanol (14 mL) was heated to reflux for 5 h. The reaction mixture wasallowed to cool to room temperature, the ethanol was removed in vacuoand the white solid collected by filtration, washing with water (3×10mL). The wet solid was dried in vacuo at 40° C. for 1 hour to yield2-(3-Bromo-pyridin-2-yl)-N-hydroxy-acetamidine as a solid (Intermediate2).

Step iii): 3-Bromo-2-(5-methyl-[1,2,4]oxadiazol-3-ylmethyl)-pyridine(Intermediate 3)

A mixture of 2-(3-bromo-pyridin-2-yl)-N-hydroxy-acetamidine obtained inStep ii) (1.24 g, 5.39 mmol) and acetic anhydride (552 mg, 5.41 mmol)were stirred in dry THF (10 mL) at 20° C. for 1.5 h. The reactionmixture was concentrated in vacuo and the residue was stirred vigorouslyin a two-phase system of 2 M Na₂CO₃ (10 mL) and DCM (10 mL) at 20° C.for 10 min. The resultant white precipitate was collected by filtration,washing with water (2×5 mL) and DCM (2×5 mL), then dried in vacuo at 40°C. for 1 h, to yield O-acetyl amidoxime as a powder.

The O-acetyl amidoxime obtained (610 mg, 2.24 mmol) and K₂CO₃ (1.56 g,11.3 mmol) were stirred vigorously in CHCl₃ (6 mL) at 60° C. for 41 h.Water (5 mL) and further CHCl₃ (5 mL) were added to the mixture withvigorous stirring. The layers were separated and the organic extractconcentrated in vacuo. Crude3-bromo-2-(5-methyl-[1,2,4]oxadiazol-3-ylmethyl)-pyridine was obtainedas a yellow liquid and used in subsequent reactions without furtherpurification (Intermediate 3).

Step iv): 3-(4-Bromo-pyrazolo[1,5-a]pyridin-2-yl)-acetamide(Intermediate 4)

The 3-bromo-2-(5-methyl-[1,2,4]oxadiazol-3-ylmethyl)-pyridine obtainedin step iii) (663 mg, 2.61 mmol) was stirred at 150° C. in toluene (5.2mL) in a sealed tube for 41 h. The reaction mixture was allowed to coolto room temperature, the toluene was removed in vacuo and the residuepurified by column chromatography (gradient: 0-10% MeOH in DCM). Thesolid obtained was triturated with diethyl ether to yield3-(4-bromo-pyrazolo[1,5-a]pyridin-2-yl)-acetamide (Intermediate 4) as asolid.

Step v): 1-(4-Bromo-2-fluoro-benzyl)-4-methanesulfonyl-piperazine(Intermediate 5)

A mixture of 4-bromo-2-fluorobenzyl bromide (30.4 g, 114 mmol, 1 eq),1-methylsulfonylpiperazine (20.0 g, 114 mmol, 1 eq) and K₂CO₃ (31.6 g,228 mmol, 2 eq) in acetonitrile (500 mL) was stirred at 40° C. for 18 h.The reaction mixture was allowed to cool to room temperature, themixture was filtered through Celite, washing with EtOAc. The filtratewas concentrated in vacuo to yield the target amine.

A mixture of 4-bromo-2-fluorobenzyl bromide (30.4 g, 114 mmol, 1 eq),1-methylsulfonylpiperazine (20.0 g, 114 mmol, 1 eq) and K₂CO₃ (31.6 g,228 mmol, 2 eq) in acetonitrile (500 mL) was stirred at 40° C. for 18 h.The reaction mixture was allowed to cool to room temperature, themixture was filtered through Celite, and washed with EtOAc. The filtratewas concentrated in vacuo to yield1-(4-Bromo-2-fluoro-benzyl)-4-methanesulfonyl-piperazine (Intermediate5).

Step vi):1-[2-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-4-methanesulfonyl-piperazine(Intermediate 6)

A mixture of the aryl bromide (20.0 g, 57 mmol, 1 eq),bis(pinacolato)diboron (16.0 g, 63 mmol, 1.1 eq), PdCl₂(dppf) (2.3 g,2.9 mmol, 0.05 eq) and potassium acetate (6.2 g, 63 mmol, 1.1 eq) indegassed dioxane (200 mL) was stirred under nitrogen in a sealed tube at90° C. for 18 h. The reaction mixture was allowed to cool to roomtemperature, the mixture was filtered through a plug of silica, andwashed with DCM. The filtrate was concentrated in vacuo. The residue wasmixed with isohexane and diethyl ether was added until the productcrystallized. The product was collected by filtration as a solid.

Alternatively, A mixture of the aryl bromide (309 g, 879.7 mmol,),bis(pinacolato)diboron (245.7 g, 967.7 mmol), PdCl₂(dppf) (35.9 g, 43.9mmol) and potassium acetate (94.9 g, 967.7 mmol) in degassed dioxane (3L) were stirred under nitrogen at 100° C. for 6 h. The reaction mixturewas allowed to cool to room temperature, the mixture was filteredthrough a plug of silica, and washed with DCM. The filtrate wasconcentrated in vacuo. DCM and hexane (2 L) were added to the viscousresidue during which the product precipitated. The suspension wasfiltered affording the desired compound.

Step vii):N-4-(3-fluoro-4-((4-(methylsulfonyl)piperazin-1-yl)methyl)phenyl)pyrazolo[1.5-a]pyridin-2-yl)acetamide(Compound 1)

3 methods were used.

Step vii): First Method

1-[2-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-4-methanesulfonyl-piperazine(26.4 g, 66 mmol, 1.1 eq) was added to a solution of aryl bromide (16.0g, 63 mmol, 1 eq) in 1,4-dioxane/water (540 mL, 4:1; v/v). Na₂CO₃ (13.4g, 126 mmol, 2.0 eq) and Pd(dppf)Cl₂ (2.6 g, 3 mmol, 0.05 eq)(dppf=1,1′-Bis(diphenylphosphino)ferrocene) were added to the degassedsolution. The resultant mixture was heated at 100° C. for 2 h. Thereaction mixture was extracted with EtOAc, dried with MgSO₄, andconcentrated in vacuo. Purification by preparative HLPC yielded thetarget compounds

Step vii): Second Method

1-[2-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-4-methanesulfonyl-piperazineobtained in step(ii) above (7.5 g, 18 mmol, 1 eq) was added to asolution of aryl bromide (4.5 g, 18 mmol, 1 eq) in 1,4-dioxane/water(150 mL, 4:1; v/v). Na₂CO₃ (3.8 g, 36 mmol, 2.0 eq) and Pd(dppf)Cl₂ (0.7g, 0.9 mmol, 5%) (dppf=1,1′-Bis(diphenylphosphino)ferrocene) were addedto the degassed solution. The resultant mixture was heated at 100° C.for 2 h. The two reaction mixtures were combined and the solvent removedin vacuo. The residue was mixed with DCM and water and the insolublematerial removed by filtration. (The insoluble material was reserved andlater re-combined with the organic residue before purification.) Theaqueous layer was separated and extracted with DCM (×3). The combinedorganics were dried (MgSO₄) and concentrated in vacuo. This residue wascombined with the previously collected insoluble material and adsorbedonto silica. Purification by column chromatography (gradient: 0-5% MeOHin EtOAc) or preparative HLPC yielded the target as a brown solid. Thebrown solid was mixed with ethanol and the suspension heated to reflux.The mixture was allowed to cool to room temperature and then furthercooled with an ice-bath. The target material was collected by filtrationand dried in vacuo.

Step vii): Third Method

A mixture of 1-N-(4-bromo-pyrazolo[1,5-a]pyridine-2-yl)-acetamide (149g, 0.586 mol) and Na₂CO₃ (124 g, 1.172 mol) in a solvent mixture of1,4-dioxane (2 L)/water (0.5 L) at room temperature was degassed withN₂. To this mixture, boronic ester (270 g, 1.15 eq) and PdCl₂(dppf) (24g, 5 mol %) were added. The reactor's jacket was heated to 100° C.,degassing with N₂ was stopped and the mixture was stirred under heatingfor 45 min. UPLC revealed complete consumption of the boronic ester. Anadditional amount of boronic ester (10 g, 0.05 eq) was added andstirring was continued at 100° C. for an additional 30 min. UPLCrevealed complete conversion and single peak of the expected product.The solvent volume was removed under reduced pressure to 0.5 L of andthe resulting suspension was left at 4° C. overnight. Water (0.5 L) wasadded to the suspension and stirred for 30 min and filtered. Theresulting cake was washed with water (0.5 L) and dried under suction for1 h. The cake was transferred to a Petri dish (425 g) and left to dry inair overnight to obtain the crude desired compound. The powder was puton a pad of silicagel and washed with DCM/MeOH (9:1 mixture). The firstfraction (2.5 L) was concentrated to dryness giving a resinous solidthat was discarded. The second fraction (10 L of DCM/MeOH 9:1 mixture,10 L of 5:1 mixture and 3 L of 3:1 mixture) was concentrated to drynessgiving the desired product. The solid was suspended in EtOH (1.1 L),heated to reflux, stirred for 30 min, cooled to 5° C., stirred for 30min and filtered. The cake was washed with cold EtOH (300 mL) andtransferred to a Petri dish giving the expected product.

The compound of the invention that has been prepared according to thesynthetic method described here above is listed in Table I below, andthe NMR spectral data of the compound of the invention is given in TableII.

TABLE I Cpd MS # Structures Name MW Mes'd 1

N-4-(3-fluoro- 4-((4-(methyl- sulfonyl)piperazin- 1-yl)methyl) phenyl)pyrazolo[1,5- a]pyridin-2- yl)acetamide 445 446

TABLE II NMR Data of Representative Compound of the invention Cpd # (δ)NMR data 1 ¹H NMR δ (ppm)(DMSO-d₆): 10.86 (1 H, s, NH), 8.59 (1 H, d,ArH), 7.65-7.48 (3 H, m, ArH), 7.36 (1 H, d, ArH), 6.99 (1 H, s, ArH),7.00-6.92 (1 H, m, ArH), 3.69 (2 H, s, CH2), 3.21-3.14 (4 H, m, CH),2.91 (3 H, s, CH3), 2.64-2.51 (4 H, m, CH), 2.11 (3H, s, CH3).

BIOLOGICAL EXAMPLES Example 1 In-Vitro Assays 1.1 JAK1 Inhibition Assay

1.1.1 JAK1 Assay polyGT Substrate

Recombinant human JAK1 catalytic domain (amino acids 866-1154; catalognumber PV4774) is purchased from Invitrogen. 25 ng of JAK1 is incubatedwith 6.25 μg polyGT substrate (Sigma catalog number P0275) in kinasereaction buffer (15 mM Hepes pH7.5, 0.01% Tween20, 10 mM MgCl₂, 2 μMnon-radioactive ATP, 0.25 μCi ³³P-gamma-ATP (Perkin Elmer, catalognumber NEG602K001MC) final concentrations) with or without 5 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 75 min at 30° C., reactions are stopped by adding of 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionis transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates are washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates is sealed. 40μL/well of Microscint-20 is added, the top of the plates was sealed anda readout was performed using the Topcount (Perkin Elmer). Kinaseactivity is calculated by subtracting counts per min (cpm) obtained inthe presence of a positive control inhibitor (10 μM staurosporine) fromcpm obtained in the presence of vehicle. The ability of a test compoundto inhibit this activity is determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK1 assay and the calculation of theIC₅₀ for the compound. The compound is tested at a concentration of 20μM followed by a ⅓ serial dilution, 8 points (20 μM-6.67 μM-2.22 μM-740nM-247 nM-82 nM-27 nM-9 nM) in a final concentration of 1% DMSO. Whencompound potency increases, more dilutions are prepared and/or the topconcentration is lowered (e.g. 5 μM, 1 μM).

1.1.2 JAK1 Ulight-JAK1 Peptide Assay

Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalognumber PV4774) was purchased from Invitrogen. 1 ng of JAK1 was incubatedwith 20 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalog numberTRF0121) in kinase reaction buffer (25 mM MOPS pH6.8, 0.01% Brij-35, 5mM MgCl₂, 2 mM DTT, 7 μM ATP) with or without 4 μL containing testcompound or vehicle (DMSO, 1% final concentration), in a total volume of20 μL, in a white 384 Opti plate (Perkin Elmer, catalog number 6007290).After 60 min at room temperature, reactions were stopped by adding 20μL/well of detection mixture (1× detection buffer (Perkin Elmer, catalognumber CR97-100C), 0.5 nM Europium-anti-phosphotyrosine (PT66) (PerkinElmer, catalog number AD0068), 10 mM EDTA). Readout is performed usingthe Envision with excitation at 320 nm and measuring emission at 615 nm(Perkin Elmer). Kinase activity was calculated by subtracting relativefluorescence units (RFU) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from RFU obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:

Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

A dose dilution series was prepared for the compound enabling thetesting of dose-response effects in the JAK1 assay and the calculationof the IC₅₀ for the compound. The compound is routinely tested at aconcentration of 20 μM followed by a ⅕ serial dilution, 10 points in afinal concentration of 1% DMSO. When compound potency increases, moredilutions are prepared and/or the top concentration is lowered (e.g. 5μM, 1 μM). The data are expressed as the average IC₅₀ from theassays±standard error of the mean.

The activity of the compound of the invention against JAK1 has beendetermined in accordance with the assay described above, thus returningIC₅₀ values of 8.18, 11.5, 7.71, 35.2, and 7.85 nM.

1.2 JAK1 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 1 ng ofJAK1 (Invitrogen, PV4774) is used in the assay. The substrate is 20 nMUlight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121). The reaction isperformed in 25 mM MOPS pH 6.8, 0.01%, Brij-35, 2 mM DTT, 5 mM MgCl₂with varying concentrations of ATP and compound. Phosphorylatedsubstrate is measured using an Europium-labeled anti-phosphotyrosineantibody PT66 (Perkin Elmer, AD0068) as described in 1.1.2. Readout isperformed on the envision (Perkin Elmer) with excitation at 320 nm andemission followed at 615 nm and 665 nm.

1.2 JAK2 Inhibition Assay

1.2.1 JAK2 Assay polyGT Substrate

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalognumber PV4210) is purchased from Invitrogen. 0.05 mU of JAK2 isincubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (10 mM MOPS pH 7.5, 0.5 mM EDTA, 0.01% Brij-35, 1mM DTT, 15 mM MgAc, 1 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP(Perkin Elmer, catalog number NEG602K001MC) final concentrations) withor without 5 μL containing test compound or vehicle (DMSO, 1% finalconcentration), in a total volume of 25 μL, in a polypropylene 96-wellplate (Greiner, V-bottom). After 90 min at 30° C., reactions are stoppedby adding of 25 μL/well of 150 mM phosphoric acid. All of the terminatedkinase reaction is transferred to prewashed (75 mM phosphoric acid) 96well filter plates (Perkin Elmer catalog number 6005177) using a cellharvester (Perkin Elmer). Plates are washed 6 times with 300 μL per wellof a 75 mM phosphoric acid solution and the bottom of the plates issealed. 40 μL/well of Microscint-20 is added, the top of the plates issealed and readout is performed using the Topcount (Perkin Elmer).Kinase activity is calculated by subtracting counts per min (cpm)obtained in the presence of a positive control inhibitor (10 μMstaurosporine) from cpm obtained in the presence of vehicle. The abilityof a test compound to inhibit this activity is determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK2 assay and the calculation of theIC₅₀ for each compound. The compound is tested at concentration of 20 μMfollowed by a ⅓ serial dilution, 8 points (20 μM-6.67 μM-2.22 μM-740nM-247 nM-82 nM-27 nM-9 nM) in a final concentration of 1% DMSO. Whenpotency of compound series increases, more dilutions are prepared and/orthe top concentration is lowered (e.g. 5 μM, 1 μM).

1.2.2 JAK2 Ulight-JAK1 Peptide Assay

Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalognumber PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 wasincubated with 25 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalognumber TRF0121) in kinase reaction buffer (25 mM HEPES pH7.0, 0.01%Triton X-100, 7.5 mM MgCl₂, 2 mM DTT, 7.5 μM ATP) with or without 4 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 20 μL, in a white 384 Opti plate (Perkin Elmer, catalognumber 6007290). After 60 min at room temperature, reactions werestopped by adding 20 μL/well of detection mixture (1× detection buffer(Perkin Elmer, catalog number CR97-100C), 0.5 nMEuropium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog numberAD0068), 10 mM EDTA). Readout is performed using the Envision withexcitation at 320 nm and measuring emission at 615 nm (Perkin Elmer).Kinase activity was calculated by subtracting relative fluorescenceunits (RFU) obtained in the presence of a positive control inhibitor (10μM staurosporine) from RFU obtained in the presence of vehicle. Theability of a test compound to inhibit this activity was determined as:

Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for compound enabling the testing ofdose-response effects in the JAK2 assay and the calculation of the IC₅₀for the compound. The compound is tested at concentration of 20 μMfollowed by a ⅕ serial dilution, 10 points in a final concentration of1% DMSO. When compound potency increases, more dilutions are preparedand/or the top concentration is lowered (e.g. 5 μM, 1 μM). The data areexpressed as the average IC₅₀ from the assays±standard error of themean.

The activity of the compound of the invention against JAK2 has beendetermined in accordance with the assay described above, thus returningIC₅₀ values of 17.3, 11.3, 9.57, 26, and 18.2 nM.

1.4 JAK2 Kd/Ki Determination Assay 1.4.1. JAK2 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 0.0125mU of JAK1 (Invitrogen, PV4210) is used in the assay. The substrate is25 nM Ulight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121). Thereaction is performed in 25 mM HEPES pH7.0, 0.01% Triton X-100, 7.5 mMMgCl₂, 2 mM DTT with varying concentrations of ATP and compound.Phosphorylated substrate is measured using a Europium-labeledanti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068) as describedin 1.2.2. Readout was performed on the envision (Perkin Elmer) withexcitation at 320 nm and emission followed at 615 nm and 665 nm.

1.4.2. JAK2 Kd Determination Assay

JAK2 (Invitrogen, PV4210) is used at a final concentration of 2.5 nM.The binding experiment is performed in 50 mM Hepes pH 7.5, 0.01%Brij-35, 10 mM MgCl₂, 1 mM EGTA using 25 nM kinase tracer 236(Invitrogen, PV5592) and 2 nM Europium-anti-GST (Invitrogen, PV5594)with varying compound concentrations. Detection of tracer is performedaccording to the manufacturer's procedure.

1.5 JAK3 Inhibition Assay

Recombinant human JAK3 catalytic domain (amino acids 795-1124; catalognumber 08-046) was purchased from Carna Biosciences. 0.5 ng JAK3 proteinwas incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275)in kinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 10 mM MgCl₂,2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mM b-glycerolphosphate, 0.01% Triton X-100,1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (Perkin Elmer, catalognumber NEG602K001MC) final concentrations) with or without 5 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 45 min at 30° C., reactions were stopped by adding 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionwas transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates was sealed. 40μL/well of Microscint-20 was added, the top of the plates was sealed andreadout was performed using the Topcount (Perkin Elmer). Kinase activitywas calculated by subtracting counts per min (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity was determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK3 assay and the calculationof the IC₅₀ for the compound. The compound was tested at concentrationof 20 μM followed by a ⅕ serial dilution, 10 points in a finalconcentration of 1% DMSO. When compound increased, more dilutions wereprepared and/or the top concentration was lowered (e.g. 5 μM, 1 μM).

The activity of the compound of the invention against JAK3 has beendetermined in accordance with the assay described above, thus returningIC₅₀ values of 271, 299, 213, and 428 nM.

1.6 JAK3 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK3(Carna Biosciences, 08-046) was used at a final concentration of 20ng/ml. The substrate was Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50000 (Sigma, P0275) The reaction was performed in 25 mM Tris pH 7.5,0.01% Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mMb-glycerolphosphate, 10 mM MgCl₂ with varying concentrations of ATP andcompound and stopped by addition of 150 mM phosphoric acid. Measurementof incorporated phosphate into the substrate polyGT was done by loadingthe samples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated ³³P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer).

For example, when tested in this assay, the compound of the inventionreturned a Ki=227 nM

1.7 TYK2 Inhibition Assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalognumber 08-147) was purchased from Carna biosciences. 4 ng of TYK2 wasincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Hepes pH 7.2, 50 mM NaCl, 0.5 mM EDTA, 1mM DTT, 5 mM MnCl₂, 10 mM MgCl₂, 0.1% Brij-35, 0.1 μM non-radioactiveATP, 0.125 μCi ³³P-gamma-ATP (Perkin Elmer, catalog number NEG602K001MC)final concentrations) with or without 5 μL containing test compound orvehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C.,reactions were stopped by adding 25 μL/well of 150 mM phosphoric acid.All of the terminated kinase reaction was transferred to prewashed (75mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number6005177) using a cell harvester (Perkin Elmer). Plates were washed 6times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates was sealed. 40 μL/well of Microscint-20 was added,the top of the plates was sealed and readout was performed using theTopcount (Perkin Elmer). Kinase activity was calculated by subtractingcounts per min (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the TYK2 assay and the calculationof the IC50 for each compound. Each compound was routinely tested atconcentration of 20 μM followed by a ⅓ serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions were prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM).

The activity of the compound of the invention against TYK2 has beendetermined in accordance with the assay described above, thus returningIC₅₀ values of 167, 135, 119, and 157 nM

1.8 TYK2 Kd/Ki Determination Assay 1.8.1 TYK2 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). TYK2(Carna Biosciences, 08-147) was used at a final concentration of 160ng/ml. The substrate was Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50000 (Sigma, P0275) The reaction was performed in 25 mM Hepes pH 7.2, 50mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM MnCl2, 10 mM MgCl₂, 0.1% Brij-35with varying concentrations of ATP and compound and stopped by additionof 150 mM phosphoric acid. Measurement of incorporated phosphate intothe substrate polyGT was done by loading the samples on a filter plate(using a harvester, Perkin Elmer) and subsequent washing. Incorporated³³P in polyGT is measured in a Topcount scintillation counter afteraddition of scintillation liquid to the filter plates (Perkin Elmer).

For example, when tested in this assay, the compound of the inventionreturned a Ki=110 nM

1.8.2 TYK2 Kd Determination Assay

TYK2 (Carna Biosciences, 08-147) is used at a final concentration of 50nM. The binding experiment is performed in 50 mM Hepes pH 7.5, 0.01%Brij-35, 10 mM MgCl₂, 1 mM EGTA using 15 nM kinase tracer 236(Invitrogen, PV5592) and 10 nM Europium-anti-GST (Invitrogen, PV5594)with varying compound concentrations. Detection of tracer is performedaccording to the manufacturers' procedure.

Example 2 Cellular Assays 2.1 JAK-STAT Signalling Assay:

HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)containing 10% heat inactivated fetal calf serum, 100 U/mL penicillinand 100 μg/mL streptomycin. HeLa cells were used at 70% confluence fortransfection. 20,000 cells in 87 μL cell culture medium were transientlytransfected with 40 ng pSTAT1(2)-luciferase reporter (Panomics), 8 ng ofLacZ reporter as internal control reporter and 52 ng of pBSK using 0.32μL Jet-PEI (Polyplus) as transfection reagent per well in 96-well plateformat. After overnight incubation at 37° C., 5% CO₂, transfectionmedium was removed. 81 μL of DMEM+1.5% heat inactivated fetal calf serumwas added. 9 μL compound at 10× concentration was added for 60 min andthen 10 μL of human OSM (Peprotech) at 33 ng/mL final concentration.

The compound was tested in duplicate starting from 20 μM followed by a ⅓serial dilution, 8 doses in total (20 μM-6.6 μM-2.2 μM-740 nM-250 nM-82nM-27 nM-9 nM) in a final concentration of 0.2% DMSO.

After overnight incubation at 37° C., 5% CO₂ cells were lysed by adding100 μL lysis buffer/well (PBS, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 10%Trehalose, 0.05% Tergitol NP9, 0.3% BSA).

40 μL of cell lysate was used to read β-galactosidase activity by adding180 μL β-Gal solution (30 μl ONPG 4 mg/mL+150 μL β-Galactosidase buffer(0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 1 mM MgCl₂)) for 20 min. The reactionwas stopped by addition of 50 μL Na₂CO₃ 1 M. Absorbance was read at 405nm.

Luciferase activity was measured using 40 μL cell lysate plus 40 μL ofSteadylite® as described by the manufacturer (Perkin Elmer), on theEnvision (Perkin Elmer).

Omitting OSM was used as a positive control (100% inhibition). Asnegative control 0.5% DMSO (0% inhibition) was used. The positive andnegative controls were used to calculate z′ and ‘percent inhibition’(PIN) values.

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

PIN values were plotted for compounds tested in dose-response and EC₅₀values were derived.

The activity of the compound of the invention against JAK-STAT has beendetermined in accordance with the assay described above, thus returningan IC₅₀ value of 970 nM.

Example 2.2 OSM/IL-1β Signaling Assay

OSM and IL-1β are shown to synergistically upregulate MMP13 levels inthe human chondrosarcoma cell line SW1353. The cells are seeded in 96well plates at 15,000 cells/well in a volume of 120 μL DMEM (Invitrogen)containing 10% (v/v) FBS and 1% penicillin/streptomycin (InVitrogen)incubated at 37° C. 5% CO₂. Cells are preincubated with 15 μL ofcompound in M199 medium with 2% DMSO 1 hr before triggering with 15 μLOSM and IL-1β to reach 25 ng/mL OSM and 1 ng/mL IL-1β, and MMP13 levelsare measured in conditioned medium 48 h after triggering. MMP13 activityis measured using an antibody capture activity assay. For this purpose,384 well plates (NUNC, 460518, MaxiSorb black) are coated with 35 μL ofa 1.5 μg/mL anti-human MMP13 antibody (R&D Systems, MAB511) solution for24 hrs at 4° C. After washing the wells 2 times with PBS+0.05% Tween,the remaining binding sites are blocked with 100 μL 5% non-fat dry milk(Santa Cruz, sc-2325, Blotto) in PBS for 24 hr at 4° C. Next, the wellsarc washed twice with PBS+0.05% Tween and 35 μL of 1/10 dilution ofculture supernatant containing MMP13 in 100-fold diluted blocking bufferis added and incubated for 4 hr at room temperature. Next the wells arewashed twice with PBS+0.05% Tween followed by MMP13 activation byaddition of 35 μL of a 1.5 mM 4-Aminophenylmercuric acetate (APMA)(Sigma, A9563) solution and incubation at 37° C. for 1 hr. The wells arewashed again with PBS+0.05% Tween and 35 μL MMP13 substrate (Biomol,P-126, OmniMMP fluorogenic substrate) is added. After incubation for 24hrs at 37° C. fluorescence of the converted substrate is measured in aPerkin Elmer Wallac EnVision 2102 Multilabel Reader (wavelengthexcitation: 320 nm, wavelength emission: 405 nm).

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

Example 2.3 PBL Proliferation Assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 andproliferation is measured using a BrdU incorporation assay. The PBL arefirst stimulated for 72 hrs with PHA to induce IL-2 receptor, then theyare fasted for 24 hrs to stop cell proliferation followed by IL-2stimulation for another 72 hrs (including 24 hr BrdU labeling). Cellsare preincubated with test compounds 1 hr before IL-2 addition. Cellsare cultured in RPMI 1640 containing 10% (v/v) FBS.

Example 2.4 Whole Blood Assay (WBA) 2.4.1 IFNα Stimulation Protocol

To predict the potency of the test compounds to inhibit JAK1 orJAK2-dependent signaling pathways in vivo, a physiologically relevant invitro model was developed using human whole blood. In the WBA assay,blood, drawn from human volunteers who gave informed consent, is treatedex vivo with compound (1 h) and subsequently stimulated either for 30min with interferon α (IFNα, JAK1 dependent pathway) or for 2 h withgranulocyte macrophage-colony stimulating factor (GM-CSF, JAK2 dependentpathway).

2.4.1.1 Phospho-STAT1 Assay

For IFNα stimulation, increase in phosphorylation of Signal Transducersand Activators of Transcription 1 (pSTAT1) by INFα in white blood cellextracts is measured using a pSTAT1 ELISA assay. Phosphorylation ofSignal Transducer and Activator of Transcription 1 (STAT1) afterinterferon alpha (IFNα) triggering is a JAK1-mediated event. ThePhospho-STAT1 Assay, which is used to measure Phospho-STAT1 levels incellular extracts, is developed to assess the ability of a compound toinhibit JAK1-dependent signaling pathways.

Whole human blood, drawn from human volunteers who gave informedconsent, is ex vivo treated with compound (1 h) and subsequentlystimulated for 30 min with IFNα. The increase in phosphorylation ofSTAT1 by INFα in white blood cell extracts was measured using aphospho-STAT1 ELISA.

The ACK lysis buffer consists of 0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA.The pH of the buffer is 7.3.

A 10× cell lysis buffer concentrate (part of the PathScan Phospho-STAT1(Tyr701) sandwich ELISA kit from Cell Signaling) is diluted 10-fold inH₂O. Proteinase inhibitors arc added to the buffer before use.

20 μg IFNα is dissolved in 40 μL H₂O to obtain a 500 μg/mL stocksolution. The stock solution is stored at −20° C.

A 3-fold dilution series of the compound is prepared in DMSO (highestconcentration: 10 mM). Subsequently, the compound is further diluted inmedium (dilution factor dependent on desired final compoundconcentration).

2.4.1.1.1 Incubation of Blood with Compound and Stimulation with IFNα

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 392 μL. Afterward, 4 μL of compound dilution is added toeach aliquot and the blood samples are incubated for 1 h at 37° C. TheIFNa stock solution is diluted 1000-fold in RPMI medium to obtain a 500ng/mL working solution. 4 μL of the 500 ng/mL work solution is added tothe blood samples (final concentration IFNα: 5 ng/ml). The samples areincubated at 37° C. for 30 min.

2.4.1.1.2 Preparation of Cell Extracts

At the end of the stimulation period, 7.6 mL ACK buffer is added to theblood samples to lyse the red blood cells. The samples are mixed byinverting the tubes five times and the reaction is incubated on ice for5 min. The lysis of the RBC should be evident during this incubation.The cells are pelleted by centrifugation at 300 g, 4° C. for 7 min andthe supernatant is removed. 10 mL 1× PBS is added to each tube and thecell pellet is resuspended. The samples are centrifuged again for 7 minat 300 g, 4° C. The supernatant is removed and the pellet resuspended in500 μL of 1× PBS. Then, the cell suspension is transferred to a clean1.5 mL microcentrifuge tube. The cells are pelleted by centrifugation at700 g for 5 min at 4° C. The supernatant is removed and the pellet isdissolved in 150 μL cell lysis buffer. The samples are incubated on icefor 15 min. After that, the samples are stored at −80° C. until furtherprocessing.

2.4.1.1.3 Measurement of STAT1 Phosphorylation by ELISA

The Pathscan Phospho-STAT1 (Tyr701) Sandwich ELISA kit from CellSignaling (Cat.no: #7234) is used to determine Phospho-STAT1 levels.

The cellular extracts are thawed on ice. The tubes are centrifuged for 5min at 16,000 g, 4° C. and the cleared lysates are harvested. Meanwhile,the microwell strips from the kit are equilibrated to room temperatureand wash buffer is prepared by diluting 20× wash buffer in H₂O. Samplesarc diluted 2-fold in sample diluent and 100 μL is added to themicrowell strips. The strips are incubated overnight at 4° C.

The following day, the wells are washed 3 times with wash buffer. 100 μLof the detection antibody is added to the wells. The strips areincubated at 37° C. for 1 h. Then, the wells are washed 3 times withwash buffer again. 100 μL HRP-linked secondary antibody is added to eachwell and the samples are incubated at 37° C. After 30 min, the wells arewashed 3 times again and 100 μL TMB substrate is added to all wells.When samples turned blue, 100 μL STOP solution is added to stop thereaction. Absorbance is measured at 450 nm.

2.4.1.2 Data Analysis

Inhibition of phosphoSTAT1 induction by IFNα in cell extracts is plottedagainst the compound concentration and IC₅₀ values are derived usingGraphpad software. Data are retained if R² is larger than 0.8 and thehill slope is smaller than 3.

2.4.1.3 IL-8 ELISA

For GM-CSF stimulation, increase in interleukin-8 (IL-8) levels inplasma is measured using an IL-8 ELISA assay. Granulocytemacrophage-colony stimulating factor (GM-CSF)—induced interleukin 8(IL-8) expression is a JAK2-mediated event. The IL-8 ELISA, which can beused to measure IL-8 levels in plasma samples, has been developed toassess the ability of a compound to inhibit JAK2-dependent signalingpathways.

Whole human blood, drawn from human volunteers who gave informedconsent, is ex vivo treated with compound (1 h) and subsequentlystimulated for 2 h with GM-CSF. The increase in IL-8 levels in plasma ismeasured using an IL-8 ELISA assay.

10 μg GM-CSF is dissolved in 100 μL H₂O to obtain a 100 μg/mL stocksolution. The stock solution is stored at −20° C.

A 3-fold dilution series of the test compound is prepared in DMSO(highest concentration: 10 mM). Subsequently, the compound is furtherdiluted in medium (dilution factor dependent on desired final compoundconcentration).

2.4.1.3.1 Incubation of Blood with Compound and Stimulation with GM-CSF

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 245 μL. Afterwards, 2.5 μL test compound dilution is addedto each aliquot and the blood samples are incubated for 1 h at 37° C.The GM-CSF stock solution is diluted 100-fold in RPMI medium to obtain a1 μg/mL work solution. 2.5 μL of the 1 μg/mL work solution is added tothe blood samples (final concentration GM-CSF: 10 ng/mL). The samplesare incubated at 37° C. for 2 h.

2.4.1.3.2 Preparation of Plasma Samples

The samples arc centrifuged for 15 min at 1,000 g, 4° C. 100 μL of theplasma is harvested and stored at −80° C. until further use.

2.4.1.3.3 Measurement of IL-8 Levels by ELISA

The Human IL-8 Chemiluminescent Immunoassay kit from R&D Systems(Cat.no: Q8000B) is used to determine IL-8 levels.

Wash buffer is prepared by diluting 10× wash buffer in H₂O. Working gloreagent is prepared by adding 1 part Glo Reagent 1 to 2 parts GloReagent B 15 min to 4 h before use.

100 μL assay diluent RD1-86 is added to each well. After that, 50 μL ofsample (plasma) is added. The ELISA plate is incubated for 2 h at roomtemperature, 500 rpm. All wells are washed 4 times with wash buffer and200 μL IL-8 conjugate is added to each well. After incubation for 3 h atroom temperature, the wells are washed 4 times with wash buffer and 100μL working glo reagent is added to each well. The ELISA plate isincubated for 5 min at room temperature (protected from light).Luminescence is measured (0.5 s/well read time).

2.4.2 IL-6 Stimulation Protocol

In addition, a flow cytometry analysis is performed to establish JAK1over JAK2 compound selectivity ex vivo using human whole blood.Therefore, blood is taken from human volunteers who gave informedconsent. Blood is then equilibrated for 30 min at 37° C. under gentlerocking, then aliquoted in Eppendorf tubes. Compound is added atdifferent concentrations and incubated at 37° C. for 30 min under gentlerocking and subsequently stimulated for 20 min at 37° C. under gentlerocking with interleukin 6 (IL-6) for JAK1-dependent pathway stimulationor GM-CSF for JAK2-dependent pathway stimulation. Phospho-STAT1 andphospho-STAT5 are then evaluated using FACS analysis.

2.4.2.1 Phospho-STAT1 Assays

For IL-6-stimulated increase of Signal Transducers and Activators ofTranscription 1 (pSTAT1) phosphorylation in white blood cell, humanwhole blood, drawn from human volunteers who gave informed consent, isex vivo treated with the compound for 30 min and subsequently stimulatedfor 20 min with IL-6. The increase in phosphorylation of STAT1 by IL-6in lymphocytes is measured using anti phospho-STAT1 antibody by FACS.

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) is diluted 5-foldwith distilled water and pre-warmed at 37° C. The remaining dilutedLyse/Fix buffer is discarded.

10 μg rhIL-6 (R&D Systems, Cat no 206-IL) is dissolved in 1 ml of PBS0.1% BSA to obtain a 10 μg/ml stock solution. The stock solution isaliquoted and stored at −80° C.

A 3-fold dilution series of the compound is prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples are incubated with a 1% final DMSO concentration.

2.4.2.1.1 Incubation of Blood with Compound and Stimulation with IL-6

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μl. Then, 1.5 μl of the test compound dilution isadded to each blood aliquot and the blood samples are incubated for 30min at 37° C. under gentle rocking. IL-6 stock solution (1.5 μl) isadded to the blood samples (final concentration 10 ng/ml) and samplesare incubated at 37° C. for 20 min under gentle rocking.

2.4.2.1.2 White Blood Cell Preparation and CD4 Labeling

At the end of the stimulation period, 3 ml of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes, then frozen at −80° C. until furtheruse.

For the following steps, tubes are thawed at 37° C. for approximately 20min and centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 ml of cold 1× PBS, and after centrifugation the cellpellet is resuspended in 100 μl of PBS containing 3% BSA.FITC-conjugated anti-CD4 antibody or control FITC-conjugated isotypeantibody are added and incubated for 20 min at room temperature, in thedark.

2.4.2.1.3 Cell Permeabilization and Labeling with Anti Phospho-STAT1Antibody

After washing cells with 1× PBS, the cell pellet is resuspended in 100μl of ice-cold 1× PBS and 900 μl ice-cold 100% methanol is added. Cellsare then incubated at 4° C. for 30 min for permeabilization.

Permeabilized cells are then washed with 1× PBS containing 3% BSA andfinally resuspended in 80 μl of 1× PBX containing 3% BSA.

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. no 612564 and 559319, respectively) areadded and mixed, then incubated for 30 min at 4° C., in the dark.

Cells are then washed once with 1× PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

2.4.2.1.4 Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT1 positive cells aremeasured after gating on CD4+ cells, in the lymphocyte gate. Data areanalyzed using the FACSDiva software and the percentage inhibition ofIL-6 stimulation calculated on the percentage of positive cells forphospho-STAT1 on CD4+ cells.

2.4.2.2 Phospho-STAT5 Assay

For GM-CSF-stimulated increase of Signal Transducers and Activators ofTranscription 5 (pSTAT5) phosphorylation in white blood cell, humanwhole blood, drawn from human volunteers who gave informed consent, isex vivo treated with compound for 30 min and subsequently stimulated for20 min with GM-CSF. The increase in phosphorylation of STATS by GM-CSFin monocytes is measured using an anti phospho-STAT5 antibody by FACS.

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) is diluted 5-foldwith distilled water and pre-warmed at 37° C. Remaining diluted Lyse/Fixbuffer is discarded.

10 μg rhGM-CSF (AbCys S.A., Cat no P300-03) is dissolved in 100 μl ofPBS 0.1% BSA to obtain a 100 μg/ml stock solution. The stock solution isstored aliquoted at −80° C.

A 3-fold dilution series of the compound is prepared in DMSO (10 mMstock solution). Control-treated samples receive DMSO without the testcompound. All samples are incubated with a 1% final DMSO concentration.

2.4.2.2.1 Incubation of Blood with Compound and Stimulation with GM-CSF

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μl. Then, 1.5 μl of compound dilution is added to eachaliquot and the blood samples are incubated for 30 min at 37° C. undergentle rocking. GM-CSF stock solution (1.5 μl) is added to the bloodsamples (final concentration 20 pg/ml) and samples are incubated at 37°C. for 20 min under gentle rocking.

2.4.2.2.2 White Blood Cell Preparation and CD14 Labeling

At the end of the stimulation period, 3 ml of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes, then frozen at −80° C. until furtheruse.

For the following steps, tubes are thawed at 37° C. for approximately 20min and centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 ml of cold 1× PBS, and after centrifugation the cellpellet is resuspended in 100 μl of PBS containing 3% BSA. FITC mouseanti-CD14 antibody (BD Biosciences, Cat. no 345784) or control FITCmouse IgG2bκ isotype antibody (BD Biosciences, Cat. no 555057) are addedand incubated for 20 min at room temperature, in the dark.

2.4.2.2.3 Cell Permeabilization and Labeling with Anti Phospho-STATSAntibody

After washing cells with 1× PBS, the cell pellet is resuspended in 100μl of ice-cold 1× PBS and 900 μl of ice-cold 100% methanol is added.Cells are then incubated at 4° C. for 30 min for permeabilization.

Permeabilized cells are then washed with 1× PBS containing 3% BSA andfinally resuspended in 80 μl of 1× PBX containing 3% BSA.

20 μL of PE mouse anti-STATS (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567and 554680, respectively) areadded, mixed then incubated for 30 min at 4° C., in the dark.

Cells are then washed once with 1× PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

2.4.2.2.4 Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT5 positive cells aremeasured after gating on CD14+ cells. Data are analyzed using theFACSDiva software and correspond to the percentage of inhibition ofGM-CSF stimulation calculated on the percentage of positive cells forphosphor-STAT5 on CD14+ cells.

Example 3 In Vivo Models Example 3.1 CIA Model 3.1.1 Materials

Complete Freund's adjuvant (CFA) and Incomplete Freund's adjuvant (IFA)are purchased from Difco. Bovine collagen type II (CII),lipopolysaccharide (LPS), and Enbrel® is obtained from Chondrex (Isled'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25 mginjectable syringe, France) Acros Organics (Palo Alto, Calif.),respectively. All other reagents used are of reagent grade and allsolvents are of analytical grade.

3.1.2 Animals

Dark Agouti rats (male, 7-8 weeks old) are obtained from HarlanLaboratories (Maison-Alfort, France). Rats are kept on a 12 hrlight/dark cycle (0700-1900). Temperature is maintained at 22° C., andfood and water are provided ad libitum.

3.1.3 Collagen Induced Arthritis (CIA)

One day before the experiment, CII solution (2 mg/mL) is prepared with0.05 M acetic acid and stored at 4° C. Just before the immunization,equal volumes of adjuvant (IFA) and CII are mixed by a homogenizer in apre-cooled glass bottle in an ice water bath. Extra adjuvant andprolonged homogenization may be required if an emulsion is not formed.0.2 mL of the emulsion is injected intradermally at the base of the tailof each rat on day 1, a second booster intradermal injection (CIIsolution at 2 mg/mL in CFA 0.1 mL saline) is performed on day 9. Thisimmunization method is modified from published methods (Sims et al,2004; Jou et al., 2005).

3.1.4 Study Design

The therapeutic effects of the compounds are tested in the rat CIAmodel. Rats are randomly divided into equal groups and each groupcontained 10 rats. All rats are immunized on day 1 and boosted on day 9.Therapeutic dosing lasts from day 16 to day 30. The negative controlgroup is treated with vehicle (MC 0.5%) and the positive control groupwith Enbrel® (10 mg/kg, 3× week., s.c.). A compound of interest istypically tested at 3 doses, e.g. 3, 10, 30 mg/kg, p.o.

3.1.5 Clinical Assessment of Arthritis

Arthritis is scored according to the method of Khachigian 2006, Lin etal 2007 and Nishida et al. 2004). The swelling of each of the four pawsis ranked with the arthritic score as follows: 0—no symptoms; 1—mild,but definite redness and swelling of one type of joint such as the ankleor wrist, or apparent redness and swelling limited to individual digits,regardless of the number of affected digits; 2—moderate redness andswelling of two or more types of joints; 3—severe redness and swellingof the entire paw including digits; 4—maximally inflamed limb withinvolvement of multiple joints (maximum cumulative clinical arthritisscore 16 per animal) (Nishida et al., 2004).

To permit the meta-analysis of multiple studies the clinical scorevalues are normalised as follows:

AUC of clinical score (AUC score): The area under the curve (AUC) fromday 1 to day 14 is calculated for each individual rat. The AUC of eachanimal is divided by the average AUC obtained for the vehicle in thestudy from which the data on that animal is obtained and multiplied by100 (i.e. the AUC was expressed as a percentage of the average vehicleAUC per study).

Clinical score increase from day 1 to day 14 (End point score): Theclinical score difference for each animal is divided by the averageclinical score difference obtained for the vehicle in the study fromwhich the data on that animal is obtained and multiplied by 100 (i.e.the difference is expressed as a percentage of the average clinicalscore difference for the vehicle per study).

3.1.6 Change in Body Weight (%) After Onset of Arthritis

Clinically, body weight loss is associated with arthritis (Shelton etal., 2005; Argiles et al., 1998; Rall, 2004; Walsmith et al., 2004).Hence, changes in body weight after onset of arthritis can be used as anon-specific endpoint to evaluate the effect of therapeutics in the ratmodel. The change in body weight (%) after onset of arthritis iscalculated as follows:

${Mice}\text{:}\mspace{14mu} \frac{{{Body}\mspace{14mu} {Weight}_{({{week}\; 6})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\; 5})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\; 5})}} \times 100\%$${Rats}\text{:}\mspace{14mu} \frac{{{Body}\mspace{14mu} {Weight}_{({{week}\; 4})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\; 3})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\; 3})}} \times 100\%$

3.1.7 Radiology

X-ray photos are taken of the hind paws of each individual animal. Arandom blind identity number is assigned to each of the photos, and theseverity of bone erosion is ranked by two independent scorers with theradiological Larsen's score system as follows: 0—normal with intact bonyoutlines and normal joint space; 1—slight abnormality with any one ortwo of the exterior metatarsal bones showing slight bone erosion;2—definite early abnormality with any three to five of the exteriormetatarsal bones showing bone erosion; 3—medium destructive abnormalitywith all the exterior metatarsal bones as well as any one or two of theinterior metatarsal bones showing definite bone erosions; 4—severedestructive abnormality with all the metatarsal bones showing definitebone erosion and at least one of the inner metatarsal joints completelyeroded leaving some bony joint outlines partly preserved; 5—mutilatingabnormality without bony outlines. This scoring system is a modificationfrom Salvemini et al., 2001; Bush et al., 2002; Sims et al., 2004; Jouet al., 2005.

3.1.8 Histology

After radiological analysis, the hind paws of mice are fixed in 10%phosphate-buffered formalin (pH 7.4), decalcified with rapid bonedecalcifiant for fine histology (Laboratories Eurobio) and embedded inparaffin. To ensure extensive evaluation of the arthritic joints, atleast four serial sections (5 μm thick) are cut and each series ofsections are 100 μm in between. The sections are stained withhematoxylin and eosin (H&E). Histologic examinations for synovialinflammation and bone and cartilage damage are performed using a doubleblind protocol. In each paw, four parameters are assessed using afour-point scale. The parameters are cell infiltration, pannus severity,cartilage erosion and bone erosion. Scoring is performed accordingly, asfollows: 1—normal, 2—mild, 3—moderate, 4—marked. The four scores aresummed together and represented as an additional score, namely the ‘RAtotal score’.

3.1.9 Micro-Computed Tomography (μCT) Analysis of Calcaneus (Heel Bone):

Bone degradation observed in RA occurs especially at the cortical boneand can be revealed by μCT analysis (Sims N A el al., Arthritis Rheum.50 (2004) 2338-2346: Targeting osteoclasts with zoledronic acid preventsbone destruction in collagen-induced arthritis; Oste L et al., ECTCMontreal 2007: A high throughput method of measuring bone architecturaldisturbance in a murine CIA model by micro-CT morphometry). Afterscanning and 3D volume reconstruction of the calcaneus bone, bonedegradation is measured as the number of discrete objects present perslide, isolated in silico perpendicular to the longitudinal axis of thebone. The more the bone is degraded, the more discrete objects aremeasured. 1000 slices, evenly distributed along the calcaneus (spaced byabout 10.8 μm), are analyzed.

3.1.10 Steady State PK

At day 7 or 11, blood samples are collected at the retro-orbital sinuswith lithium heparin as anti-coagulant at the following time points:predose, 1, 3 and 6 hrs. Whole blood samples are centrifuged and theresulting plasma samples are stored at −20° C. pending analysis. Plasmaconcentrations of each test compound are determined by an LC-MS/MSmethod in which the mass spectrometer is operated in positiveelectrospray mode. Pharmacokinetic parameters are calculated usingWinnonlin® (Pharsight®, United States) and it is assumed that thepredose plasma levels are equal to the 24 hrs plasma levels.

3.1.11 Results

The compound of the invention exhibited statistically significantimprovements in the normalized clinical score values (calculated as AUCor as the difference from day 1 to day 14) at a dose of 0.1 mg/kg.

Example 3.2 Septic Shock Model

Injection of lipopolysaccharide (LPS) induces a rapid release of solubletumour necrosis factor (TNF-alpha) into the periphery. This model isused to analyse prospective blockers of TNF release in vivo.

Six BALB/cJ female mice (20 g) per group are treated at the intendeddosing once, po. Thirty min later, LPS (15 μg/kg; E. Coli serotype0111:B4) is injected ip. Ninety min later, mice are euthanized and bloodis collected. Circulating TNF alpha levels are determined usingcommercially available ELISA kits. Dexamethasone (5 μg/kg) is used as areference anti-inflammatory compound.

Example 3.3 MAB Model

The MAB model allows a rapid assessment of the modulation of an RA-likeinflammatory response by therapeutics (Kachigian L M. Nature Protocols(2006) 2512-2516: Collagen antibody-induced arthritis). DBA/J mice areinjected i.v. with a cocktail of mAbs directed against collagen II. Oneday later, compound treatment is initiated (vehicle: 10% (v/v) HPβCD).Three days later, mice receive an i.p. LPS injection (50 μg/mouse),resulting in a fast onset of inflammation. Compound treatment iscontinued until 10 days after the mAb injection. Inflammation is read bymeasuring paw swelling and recording the clinical score of each paw. Thecumulative clinical arthritis score of four limbs is presented to showthe severity of inflammation. A scoring system is applied to each limbusing a scale of 0-4, with 4 being the most severe inflammation.

-   -   0 Symptom free    -   1 Mild, but definite redness and swelling of one type of joint        such as the ankle or wrist, or apparent redness and swelling        limited to individual digits, regardless of the number of        affected digits    -   2 Moderate redness and swelling of two or more types of joints    -   3 Severe redness and swelling of the entire paw including digits    -   4 Maximally inflamed limb with involvement of multiple joints

Example 3.4 Oncology Models

In vivo models to validate efficacy of small molecules towardsJAK2-driven myleoproliferative diseases are described by Wernig et al.Cancer Cell 13, 311, 2008 and Geron et al. Cancer Cell 13, 321, 2008.

Example 3.5 Mouse IBD Model

In vitro and in vivo models to validate efficacy of small moleculestowards IBD are described by Wirtz et al. 2007.

Example 3.6 Mouse Asthma Model

In vitro and in vivo models to validate efficacy of small moleculestowards asthma arc described by Nials et al., 2008; Ip et al. 2006;Pernis et al., 2002; Kudlacz et al., 2008.

Example 4 Pharmacokinetic, DMPK and Toxicity Assays Example 4.1Thermodynamic Solubility

A solution of 1 mg/mL of the test compound is prepared in a 0.2Mphosphate buffer pH 7.4 or a 0.1M citrate buffer pH 3.0 at roomtemperature in a glass vial.

The samples are rotated in a Rotator drive STR 4 (Stuart Scientific,Bibby) at speed 3.0 at room temperature for 24 hrs.

After 24 hrs, 800 μL of the sample is transferred to an eppendorf tubeand centrifuged 5 min at 14000 rpm. 200 μL of the supernatant of thesample is then transferred to a MultiscreenR Solubility Plate(Millipore, MSSLBPC50) and the supernatant is filtered (10-12″ Hg) withthe aid of a vacuum manifold into a clean Greiner polypropylene V-bottom96 well plate (Cat no.651201). 5 μL of the filtrate is diluted into 95μL (F20) of the same buffer used to incubate in the plate containing thestandard curve (Greiner, Cat no. 651201).

The standard curve for the compound is prepared freshly in DMSO startingfrom a 10 mM DMSO stock solution diluted factor 2 in DMSO (5000 μM) andthen further diluted in DMSO up to 19.5 μM. 3 μl of the dilution seriesas from 5000 μM is then transferred to a 97 μL acetonitrile-buffermixture (50/50). The final concentration range is 2.5 to 150 μM.

The plate is sealed with sealing mats (MA96RD-04S, Kinesis, Cambs, PE198YX, UK) and samples are measured at room temperature on LCMS (ZQ 1525from Waters) under optimized conditions using Quanoptimize to determinethe appropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Ais 15 mM ammonia and solvent B is acetonitrile. The sample is run underpositive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column, fromWaters. The solvent gradient has a total run time of 2 min and rangesfrom 5% B to 95% B.

Peak areas are analyzed with the aid of Masslynx software package andpeak areas of the samples are plotted against the standard curve toobtain the solubility of the compound.

Solubility values are reported in μM or μg/mL.

Example 4.2 Aqueous Solubility 4.2.1 Aqueous Solubility 2% DMSOProcedure

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.2M phosphate buffer pH7.4or 0.1M citrate buffer pH 3.0 at room temperature is added.

The final concentration ranges from 200 μM to 2.5 μM in 5 equal dilutionsteps. The final DMSO concentration does not exceed 2%. 200 μM Pyrene isadded to the corner points of each 96 well plate and serves as areference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 hr at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwhich is plotted onto a graph. The first concentration at which thecompound appears completely dissolved is the concentration that isreported below, however the true concentration will lie somewherebetween this concentration and one dilution step higher.

Solubility values mesured according to this protocol are reported inμg/mL.

4.2.2 Aqueous Solubility 3% DMSO Procedure

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.1M phosphate buffer pH7.4or 0.1M citrate buffer pH3.0 at room temperature is added.

The final concentration will range from 300 μM to 18.75 μM in 5 equaldilution steps. The final DMSO concentration does not exceed 3%. 200 μMPyrene is added to the corner points of each 96 well plate and serves asa reference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 h at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwith a software tool which can be plotted onto a graph. The firstconcentration at which the compound appears completely dissolved is theconcentration reported; however the true concentration lies somewherebetween this concentration and one dilution step higher.

Solubility values mesured according to this protocol are reported inμg/mL.

Example 4.3 Plasma Protein Binding (Equilibrium Dialysis)

A 10 mM stock solution of the compound in DMSO is diluted with a factor5 in DMSO. This solution is further diluted in freshly thawed human,rat, mouse or dog plasma (BioReclamation INC) with a final concentrationof 10 [M and final DMSO concentration of 0.5% (5.5 μL in 1094.5 μLplasma in a PP-Masterblock 96 well (Greiner, Cat no. 780285))

A Pierce Red Device plate with inserts (ThermoScientific, Cat no. 89809)is prepared and filled with 750 μL PBS in the buffer chamber and 500 μLof the spiked plasma in the plasma chamber. The plate is incubated for 4hrs at 37° C. while shaking at 230 rpm. After incubation, 120 μL of bothchambers is transferred to 360 μL acetonitrile in a 96-well roundbottom, PP deep-well plates (Nunc, Cat no. 278743) and sealed with analuminum foil lid. The samples are mixed and placed on ice for 30 min.This plate is then centrifuged 30 min at 1200 rcf at 4° C. and thesupernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201)for analysis on LCMS.

The plate is sealed with sealing mats (MA96RD-04S) of Kinesis, Cambs,PE19 8YX, UK and samples arc measured at room temperature on LCMS (ZQ1525 from Waters) under optimized conditions using Quanoptimize todetermine the appropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 ml/min. Solvent Ais 15 mM ammonia and solvent B is acetonitrile. The sample is run underpositive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column, fromWaters. The solvent gradient has a total run time of 2 min and rangesfrom 5% B to 95% B.

Peak area from the compound in the buffer chamber and the plasma chamberare considered to be 100% compound. The percentage bound to plasma isderived from these results and is reported as percentage bound toplasma.

The solubility of the compound in the final test concentration in PBS isinspected by microscope to indicate whether precipitation is observed ornot.

Example 4.4 Microsomal Stability 4.4.1 Microsomal Stability 1 hIncubation Procedure

A 10 mM stock solution of compound in DMSO is diluted 1000 fold in a 182mM phosphate buffer pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-incubated at 37° C.

40 μL of deionised water is added to a well of a polypropylene Matrix 2Dbarcode labelled storage tube (Thermo Scientific) and pre-incubated at37° C.

A Glucose-6-phophate-dehydrogenase (G6PDH) working stock solution isprepared in 182 mM phosphate buffer pH7.4 and placed on ice before use.A co-factor containing MgCl₂, glucose-6-phosphate and NADP+ is preparedin deionised water and placed on ice before use.

A final working solution containing liver microsomes (Xenotech) of aspecies of interest (human, mouse, rat, dog), previously described G6PDHand co-factors is prepared and this mix is incubated for no longer than20 min at room temperature.

30 μL of the pre-heated compound dilution is added to 40 μL ofpre-heated water in the Matrix tubes and 30 μL of the microsomal mix isadded. Final reaction concentrations are 3 μM compound, 1 mg microsomes,0.4 U/mL GDPDH, 3.3 mM MgCl₂, 3.3 mM glucose-6-phosphate and 1.3 mMNADP+.

To measure percentage remaining of compound at time zero MeOH or ACN isadded (1:1) to the well before adding the microsomal mix. The plates aresealed with Matrix Sepra seals™ (Matrix, Cat. No. 4464) and shaken for afew seconds ensure complete mixing of all components.

The samples which are not stopped are incubated at 37° C., 300 rpm andafter 1 hr of incubation the reaction is stopped with MeOH or ACN (1:1).

After stopping the reaction the samples are mixed and placed on ice for30 min to precipitate the proteins. The plates are then centrifuged 30min at 1200 ref at 4° C. and the supernatant is transferred to a 96v-bottom PP plate (Greiner, 651201) for analysis on LCMS.

These plates are sealed with sealing mats (MA96RD-04S) of Kinesis,Cambs, PE19 8YX, UK and samples arc measured at room temperature on LCMS(ZQ 1525 from Waters) under optimized conditions using Quanoptimize todetermine the appropriate mass of the parent molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min Solvent Ais 15 mM ammonia and solvent B is methanol or acetonitrile, depending onthe stop solution used. The samples are run under positive ion spray onan XBridge C18 3.5 μM (2.1×30 mm) column, from Waters. The solventgradient has a total run time of 2 min and ranges from 5% B to 95% B.

Peak area from the parent compound at time 0 is considered to be 100%remaining The percentage remaining after 1 hr incubation is calculatedfrom time 0 and is calculated as the percentage remaining. Thesolubility of the compound in the final test concentration in buffer isinspected by microscope and results are reported.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 60 min.

4.4.2 Microsomal Stability 30 mn Incubation Procedure

A 10 mM stock solution of compound in DMSO is diluted to 6 μM in a 105mM phosphate buffer, pH 7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-warmed at 37° C.

A Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) workingstock solution of 700 U/ml is diluted with a factor 1:700 in a 105 mMphosphate buffer, pH7.4. A co-factor mix containing 0.528M MgCl₂.6H₂O(Sigma, M2670), 0.528M glucose-6-phosphate (Sigma, G-7879) and 0.208MNADP+ (Sigma, N-0505) is diluted with a factor 1:8 in a 105 mM phosphatebuffer, pH7.4.

A working solution is made containing 1 mg/ml liver microsomes(Provider, Xenotech) of the species of interest (human, mouse, rat, dog. . . ), 0.8 U/ml G6PDH and co-factor mix (6.6 mM MgCl₂, 6.6 mMglucose-6-phosphate, 2.6 mM NADP+). This mix is pre-incubated for 15min, but never more than 20 min, at room temperature.

After pre-incubation, compound dilution and the mix containing themicrosomes, are added together in equal amount and incubated for 30 minat 300 rpm. For the time point of 0 min, two volumes of methanol areadded to the compound dilution before the microsome mix is added. Thefinal concentration during incubation are: 3 μM test compound or controlcompound, 0.5 mg/ml microsomes, 0.4 U/ml G6PDH, 3.3 mM MgCl₂, 3.3 mMglucose-6-phosphate and 1.3 mM NaDP+.

After 30 min of incubation, the reaction is stopped with 2 volumes ofmethanol.

Of both time points, samples are mixed, centrifuged and the supernatantis harvested for analysis on LC-MS/MS. The instrument responses (i.e.peak heights) are referenced to the zero time-point samples (as 100%) inorder to determine the percentage of compound remaining Standardcompounds Propanolol and Verapamil are included in the assay design.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 30 min

Example 4.5 Caco2 Permeability

Bi-directional Caco-2 assays arc performed as described below. Caco-2cells arc obtained from European Collection of Cell Cultures (ECACC, cat86010202) and used after a 21 day cell culture in 24-well Transwellplates (Fisher TKT-545-020B).

2×10⁵ cells/well are seeded in plating medium consisting ofDMEM+GlutaMAXI+1% NEAA+10% FBS (FetalClone II)+1% Pen/Strep. The mediumis changed every 2-3 days.

Test and reference compounds (propranolol and rhodamine123 orvinblastine, all purchased from Sigma) are prepared in Hanks' BalancedSalt Solution containing 25 mM HEPES (pH7.4) and added to either theapical (125 μL) or basolateral (600 μL) chambers of the Transwell plateassembly at a concentration of 10 μM with a final DMSO concentration of0.25%.

50 μM Lucifer Yellow (Sigma) is added to the donor buffer in all wellsto assess integrity of the cell layers by monitoring Lucifer Yellowpermeation. As Lucifer Yellow (LY) cannot freely permeate lipophilicbarriers, a high degree of LY transport indicates poor integrity of thecell layer.

After a 1 hr incubation at 37° C. while shaking at an orbital shaker at150 rpm, 70 μL aliquots are taken from both apical (A) and basal (B)chambers and added to 100 μLl 50:50 acetonitrile:water solutioncontaining analytical internal standard (0.5 μM carbamazepine) in a 96well plate.

Lucifer yellow is measured with a Spectramax Gemini XS (Ex 426 nm and Em538 nm) in a clean 96 well plate containing 150 μL of liquid frombasolateral and apical side.

Concentrations of compound in the samples are measured by highperformance liquid-chromatography/mass spectroscopy (LC-MS/MS).

Apparent permeability (P_(app)) values are calculated from therelationship:

P _(app)=[compound]_(acceptor final) ×V_(acceptor)/([compound]_(donor initial) ×V _(donor))/T _(inc) ×V_(donor)/surface area×60×10⁻⁶ cm/s

-   -   V=chamber volume    -   T_(inc)=incubation time.    -   Surface area=0.33 cm²

The Efflux ratios, as an indication of active efflux from the apicalcell surface, are calculated using the ratio of P_(app) B>A/P_(app) A>B.

The following assay acceptance criteria are used:

Propranolol: P_(app) (A>B) value≧20(×10⁻⁶ cm/s)

Rhodamine 123 or Vinblastinc: P_(app) (A>B) value<5 (×10⁻⁶ cm/s) withEfflux ratio≧5.

Lucifer yellow permeability: ≦100 nm/s

Example 4.6 Pharmacokinetic Study in Rodents 4.6.1 Animals

Sprague-Dawley rats (male, 5-6 weeks old) are obtained from Janvier(France). Rats are acclimatized for at least 7 days before treatment andwere kept on a 12 hr light/dark cycle (0700-1900). Temperature ismaintained at approximately 22° C., and food and water are provided adlibitum. Two days before administration of the test compounds, ratsundergo surgery to place a catheter in the jugular vein under isofluraneanesthesia. After the surgery, rats arc housed individually. Rats arcdeprived of food for at least 16 h before oral dosing and 6 h after.Water is provided ad libitum.

4.6.2 Pharmacokinetic Study

Compounds are formulated in PEG200/physiological saline (60/40) for theintravenous route and in 0.5% methylcellulose and 10%hydroxylpropyl-β-cyclodextrine pH3 for the oral route. Test compoundsare orally dosed as a single esophageal gavage at 5 mg/kg under a dosingvolume of 5 ml/kg and intravenously dosed as a bolus via the caudal veinat 1 mg/kg under a dosing volume of 5 mL/kg. Each group consisted of 3rats. Blood samples are collected via the jugular vein with lithiumheparin as anti-coagulant at the following time points: 0.05, 0.25, 0.5,1, 3, 5 and 8 hrs (intravenous route), and 0.25, 0.5, 1, 3, 5, 8 and 24hrs (oral route). Whole blood samples are centrifuged at 5000 rpm for 10min and the resulting plasma samples are stored at −20° C. pendinganalysis.

4.6.3 Quantification of Compound Levels in Plasma

Plasma concentrations of each test compound are determined by anLC-MS/MS method in which the mass spectrometer is operated in positiveelectrospray mode.

4.6.4 Determination of Pharmacokinetic Parameters

Pharmacokinetic parameters may be calculated using Winnonlin®(Pharsight®, United States).

Example 4.7 7-Day Rat Toxicity Study

A 7-day oral toxicity study with test compounds is performed inSprague-Dawley male rats to assess their toxic potential andtoxicokinetics, at daily doses of 100, 300 and 500 mg/kg/day, by gavage,at the constant dosage-volume of 5 mL/kg/day.

The test compounds are formulated in 30% (v/v) HPfCD in purified water.Each group includes 5 principal male rats as well as 3 satellite animalsfor toxicokinetics. A fourth group is given 30% (v/v) HPI3CD in wateronly, at the same frequency, dosage volume and by the same route ofadministration, and acted as the vehicle control group.

The goal of the study is to determine the lowest dose that resulted inno adverse events being identified (no observable adverse effectlevel—NOAEL).

Example 4.8 Hepatocyte Stability

Models to evaluate metabolic clearance in hepatocyte are described byMcGinnity et al. Drug Metabolism and Disposition 2008, 32, 11, 1247.

Example 4.9 Liability for QT Prolongation

Potential for QT prolongation is assessed in the hERG patch clamp assay.

Conventional Whole-Cell Patch-Clamp

Whole-cell patch-clamp recordings are performed using an EPC10 amplifiercontrolled by Pulse v8.77 software (HEKA). Series resistance istypically less than 10 MΩ and compensated by greater than 60%,recordings are not leak subtracted. Electrodes are manufactured fromGC150TF pipette glass (Harvard).

The external bathing solution contains: 135 mM NaCl, 5 mM KCl, 1.8 mMCaCl₂, 5 mM Glucose, 10 mM HEPES, pH 7.4.

The internal patch pipette solution contains: 100 mM Kgluconate, 20 mMKCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Na₂ATP, 2 mM Glutathione, 11 mM EGTA,10 mM HEPES, pH 7.2.

Drugs are perfused using a Biologic MEV-9/EVH-9 rapid perfusion system.

All recordings are performed on HEK293 cells stably expressing hERGchannels. Cells are cultured on 12 mm round coverslips (German glass,Bellco) anchored in the recording chamber using two platinum rods(Goodfellow). hERG currents are evoked using an activating pulse to +40mV for 1000 ms followed by a tail current pulse to −50 mV for 2000 ms,holding potential is −80 mV. Pulses are applied every 20 s and allexperiments are performed at room temperature.

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All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

It will be appreciated by those skilled in the art that the foregoingdescriptions arc exemplary and explanatory in nature, and intended toillustrate the invention and its preferred embodiments. From theforegoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art, and may be made without departing from the spirit of theinvention. All such modifications coming within the scope of theappended claims are intended to be included therein.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

At least some of the chemical names of compound of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using either ^(ChemDraw)®or TSTS®/DRAW. Any open valency appearing on a carbon, oxygen ornitrogen atom in the structures herein indicates the presence of ahydrogen atom. Where a chiral center exists in a structure but nospecific stereochemistry is shown for the chiral center, bothenantiomers associated with the chiral structure are encompassed by thestructure.

What is claimed is:
 1. The compound according to Formula I:

or a pharmaceutically acceptable salt, or a solvate, or a solvate of thepharmaceutically acceptable salt, or a biologically active metabolitethereof.
 2. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a pharmaceutically effective amount of thecompound or pharmaceutically acceptable salt thereof, according toclaim
 1. 3. The pharmaceutical composition of claim 2, wherein thecarrier is selected from a parenteral carrier, an oral carrier and atopical carrier.
 4. The pharmaceutical composition according to claim 2comprising a further therapeutic agent.
 5. A method for the treatment,or prophylaxis of allergy, inflammatory conditions, autoimmune diseases,proliferative diseases, transplant rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or interferons,comprising administering an amount of a compound or pharmaceuticallyacceptable salt thereof according to claim 1, sufficient to effect saidtreatment, or prophylaxis.
 6. The method according to claim 5, whereinthe compound or pharmaceutically acceptable salt thereof, isadministered in combination with a further therapeutic agent.
 7. Amethod for the treatment, or prophylaxis of allergy, inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons, comprising administering an amountof the pharmaceutical composition of claim 2, sufficient to effect saidtreatment, or prophylaxis.
 8. The method according to claim 7, whereinthe pharmaceutical composition is administered in combination with afurther therapeutic agent.
 9. The method according to claim 8, whereinthe further therapeutic agent is an agent for the treatment, orprophylaxis of allergy, inflammatory conditions, autoimmune diseases,proliferative diseases, transplant rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or interferons.10. The pharmaceutical composition according to claim 2 comprising afurther therapeutic agent selected from arthritis treatment agents. 11.The pharmaceutical composition according to claim 10, wherein thefurther therapeutic agent is an agent selected from analgesics, NSAIDS(non-steroidal anti-inflammatory drugs), and DMARDS (disease-modifyinganti-rheumatic drugs).
 12. A method for the treatment of rheumatoidarthritis, comprising administering an amount of a compound of claim 1,sufficient to effect said treatment.
 13. The method according to claim12, wherein said compound is administered in combination with one ormore further therapeutic agents selected from arthritis treatmentagents.
 14. The method according to claim 12, wherein said compound isadministered in combination with one further therapeutic agent selectedfrom arthritis treatment agents.
 15. A method for the treatment ofrheumatoid arthritis, comprising administering an amount of apharmaceutical composition of claim 2, sufficient to effect saidtreatment.
 16. A method for the treatment of rheumatoid arthritis,comprising administering an amount of a pharmaceutical composition ofclaim 4, sufficient to effect said treatment.
 17. The method accordingto claim 15, wherein said pharmaceutical composition is administered incombination with one or more further therapeutic agents selected fromarthritis treatment agents.
 18. The method according to claim 15,wherein said pharmaceutical composition is administered in combinationwith one further therapeutic agent selected from arthritis treatmentagents.
 19. The method according to claim 16, wherein the furthertherapeutic agent is selected from arthritis treatment agents.
 20. Themethod according to claim 13, wherein the further therapeutic agent isan agent selected from analgesics, NSAIDS (non-steroidalanti-inflammatory drugs), and DMARDS (disease-modifying anti-rheumaticdrugs).
 21. The method according to claim 14, wherein the furthertherapeutic agent is an agent selected from analgesics, NSAIDS(non-steroidal anti-inflammatory drugs), and DMARDS (disease-modifyinganti-rheumatic drugs).
 22. The method according to claim 17, wherein thefurther therapeutic agent is an agent selected from analgesics, NSAIDS(non-steroidal anti-inflammatory drugs), and DMARDS (disease-modifyinganti-rheumatic drugs).
 23. The method according to claim 18, wherein thefurther therapeutic agent is an agent selected from analgesics, NSAIDS(non-steroidal anti-inflammatory drugs), and DMARDS (disease-modifyinganti-rheumatic drugs).
 24. The method according to claim 19, wherein thefurther therapeutic agent is an agent selected from analgesics, NSAIDS(non-steroidal anti-inflammatory drugs), and DMARDS (disease-modifyinganti-rheumatic drugs).